Transient protein expression methods

ABSTRACT

Described is a method for producing a protein of interest, the method comprising: a) providing a recombinant adenoviral vector comprising nucleic acid encoding the protein of interest under control of a promoter, wherein the adenoviral vector has deletions in a first region and in a second region of the adenovirus genome, wherein each of the first region and the second region is required for adenoviral genome replication and/or adenovirus particle formation, b) propagating the adenoviral vector in a first type of complementing cells that express proteins from the first and from the second region of the adenovirus genome so as to complement the deletions of the recombinant adenoviral vector, to obtain recombinant adenovirus particles, c) infecting a culture of a second type of complementing cells with the recombinant adenovirus particles, wherein the second type of complementing cells express protein from the first region of the adenovirus genome but not protein from the second region of the adenovirus genome, to produce the protein of interest, and d) harvesting the protein of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/592,409, filed Nov. 3, 2006 now U.S. Pat. No. 7,470,523, thecontents of the entirety of which is incorporated by this reference,which is a divisional of U.S. patent application Ser. No. 10/234,007,filed Sep. 3, 2002, now U.S. Pat. No. 7,132,280, issued Nov. 7, 2006,the contents of the entirety of which, including its sequence listing,is incorporated by this reference, which is a divisional of U.S. patentapplication Ser. No. 09/549,463, filed Apr. 14, 2000, now U.S. Pat. No.6,855,544, issued Feb. 15, 2005, the entire contents of which, includingits sequence listing, is incorporated by this reference, whichapplication claims priority under 35 U.S.C. Section 119(e) toProvisional Patent Application Ser. No. 60/129,452 filed Apr. 15, 1999.

TECHNICAL FIELD

The invention relates generally to biotechnology and recombinant proteinproduction, more particularly to the use of a human cell for theproduction of proteins. The invention is particularly useful for theproduction of proteins that benefit from post-translational orperi-translational modifications such as glycosylation and properfolding.

BACKGROUND

The expression of human recombinant proteins in heterologous cells hasbeen well documented. Many production systems for recombinant proteinshave become available, ranging from bacteria, yeasts, and fungi toinsect cells, plant cells and mammalian cells. However, despite thesedevelopments, some production systems are still not optimal, or are onlysuited for production of specific classes of proteins. For instance,proteins that require post- or peri-translational modifications such asglycosylation, γ-carboxylation, or hydroxylation cannot be produced inprokaryotic production systems. Another well-known problem withprokaryotic expression systems is the incorrect folding of the productto be produced, even leading to insoluble inclusion bodies in manycases.

Eukaryotic systems are an improvement in the production of, inparticular, eukaryote derived proteins, but the available productionsystems still suffer from a number of drawbacks. The hypermannosylationin, for instance, yeast strains affects the ability of yeasts toproperly express glycoproteins. Hypermannosylation often even leads toimmune reactions when a therapeutic protein thus prepared isadministered to a patient. Furthermore, yeast secretion signals aredifferent from mammalian signals, leading to a more problematictransport of mammalian proteins, including human polypeptides, to theextracellular, which in turn results in problems with continuousproduction and/or isolation. Mammalian cells are widely used for theproduction of such proteins because of their ability to performextensive post-translational modifications. The expression ofrecombinant proteins in mammalian cells has evolved dramatically overthe past years, resulting in many cases in a routine technology.

In particular, Chinese hamster ovary cells (“CHO cells”) have become aroutine and convenient production system for the generation ofbiopharmaceutical proteins and proteins for diagnostic purposes. Anumber of characteristics make CHO cells very suitable as a host cell.The production levels that can be reached in CHO cells are extremelyhigh. The cell line provides a safe production system, which is free ofinfectious or virus-like particles. CHO cells have been extensivelycharacterized, although the history of the original cell line is vague.CHO cells can grow in suspension until reaching high densities inbioreactors, using serum-free culture media; a dhfr-mutant of CHO cells(DG-44 clone, Urlaub et al., 1983) has been developed to obtain an easyselection system by introducing an exogenous dhfr gene and thereafter awell-controlled amplification of the dhfr gene and the transgene usingmethotrexate.

However, glycoproteins or proteins comprising at least two (different)subunits continue to pose problems. The biological activity ofglycosylated proteins can be profoundly influenced by the exact natureof the oligosaccharide component. The type of glycosylation can alsohave significant effects on immunogenicity, targeting andpharmacokinetics of the glycoprotein. In recent years, major advanceshave been made in the cellular factors that determine the glycosylation,and many glycosyl transferase enzymes have been cloned. This hasresulted in research aimed at metabolic engineering of the glycosylationmachinery (Fussenegger et al., 1999; Lee et al., 1989; Vonach et al.,1998; Jenikins et al., 1998; Zhang et al., 1998; Muchmore et al., 1989).Examples of such strategies are described herein.

CHO cells lack a functional α-2,6 sialyl-transferase enzyme, resultingin the exclusive addition of sialyc acids to galactose via α-2,3linkages. It is known that the absence of α-2,6 linkages can enhance theclearance of a protein from the bloodstream. To address this problem,CHO cells have been engineered to resemble the human glycan profile bytransfecting the appropriate glycosyl transferases. CHO cells are alsoincapable of producing Lewis X oligosaccharides. CHO cell lines havebeen developed that express human N-acetyl-D-glucosaminyltransferase andα-1,3-fucosyl-transferase III. In contrast, it is known that rodentcells, including CHO cells, produce CMP-N-acetylneuraminic acidhydrolase which lead to CMP-N-acetylneuraminic acids (Jenkins et al.,1996), an enzyme that is absent in humans. The proteins that carry thistype of glycosylation can produce a strong immune response when injected(Kawashima et al., 1993). The recent identification of the rodent genethat encodes the hydrolase enzyme will most likely facilitate thedevelopment of CHO cells that lack this activity and will avoid thisrodent-type modification.

Thus, it is possible to alter the glycosylation potential of mammalianhost cells by expression of human glucosyl transferase enzymes. Yet,although the CHO-derived glycan structures on the recombinant proteinsmay mimic those present on their natural human counterparts, a potentialproblem exists in that they are still found to be far from identical.Another potential problem is that not all glycosylation enzymes havebeen cloned and are, therefore, available for metabolic engineering. Thetherapeutic administration of proteins that differ from their naturalhuman counterparts may result in activation of the immune system of thepatient and cause undesirable responses that may affect the efficacy ofthe treatment. Other problems using non-human cells may arise fromincorrect folding of proteins that occurs during or after translation,which might be dependent on the presence of the different availablechaperone proteins. Aberrant folding may occur, leading to a decrease orabsence of biological activity of the protein. Furthermore, thesimultaneous expression of separate polypeptides that will together formproteins comprised of the different subunits, like monoclonalantibodies, in correct relative abundancies is of great importance.Human cells will be better capable of providing all necessary facilitiesfor human proteins to be expressed and processed correctly.

It would thus be desirable to have methods for producing humanrecombinant proteins that involve a human cell that provides consistenthuman-type processing like post-translational and peri-translationalmodifications, such as glycosylation, which preferably is also suitablefor large-scale production.

SUMMARY OF THE INVENTION

Described are, among other things, methods and compositions forproducing recombinant proteins in a human cell line. The methods andcompositions are particularly useful for generating stable expression ofhuman recombinant proteins of interest that are modifiedpost-translationally, for example, by glycosylation. Such proteins arebelieved to have advantageous properties in comparison with theircounterparts produced in non-human systems such as Chinese hamster ovarycells.

Provided are methods for producing at least one proteinaceous substancein a cell including a eukaryotic cell having a sequence encoding atleast one adenoviral E1 protein or a functional homologue, fragmentand/or derivative thereof in its genome, which cell does not encode astructural adenoviral protein from its genome or a sequence integratedtherein, the method including providing the cell with a gene encoding arecombinant proteinaceous substance, culturing the cell in a suitablemedium and harvesting at least one proteinaceous substance from the celland/or the medium. A proteinaceous substance is a substance including atleast two amino-acids linked by a peptide bond. The substance mayfurther include one or more other molecules physically linked to theamino acid portion or not. Non-limiting examples of such other moleculesinclude carbohydrate and/or lipid molecules.

A nucleic acid sequence encoding an adenovirus structural protein shouldnot be present in the genome of the cell line for a number of reasons.One reason is that the presence of an adenoviral structural protein in apreparation of produced protein is highly undesired in many applicationsof such produced protein. Removal of the structural protein from theproduct is best achieved by avoiding its occurrence in the preparation.Preferably, the eukaryotic cell is a mammalian cell. In a preferredembodiment, the proteinaceous substance harvested from the cell and thecell itself is derived from the same species. For instance, if theprotein is intended to be administered to humans, it is preferred thatboth the cell and the proteinaceous substance harvested from the cellare of human origin. One advantage of a human cell is that most of thecommercially most attractive proteins are human.

The proteinaceous substance harvested from the cell can be anyproteinaceous substance produced by the cell. In certain embodiments, atleast one of the harvested proteinaceous substances is encoded by thegene. In other embodiments, a gene is provided to the cell to enhanceand/or induce expression of one or more endogenously present genes in acell, for instance, by providing the cell with a gene encoding a proteinthat is capable of enhancing expression of a proteinaceous substance inthe cell.

As used herein, a “gene” is a nucleic acid sequence including a nucleicacid sequence of interest in an expressible format, such as anexpression cassette. The nucleic acid sequence of interest may beexpressed from the natural promoter or a derivative thereof or anentirely heterologous promoter. The nucleic acid sequence of interestcan include introns or not. Similarly, it may be a cDNA or cDNA-likenucleic acid. The nucleic acid sequence of interest may encode aprotein. Alternatively, the nucleic acid sequence of interest can encodean anti-sense RNA.

Further provided is a method for producing at least one humanrecombinant protein in a cell, including providing a human cell having asequence encoding at least an immortalizing E1 protein of an adenovirusor a functional derivative, homologue or fragment thereof in its genome,which cell does not produce structural adenoviral proteins, with anucleic acid encoding the human recombinant protein. The method involvesculturing the cell in a suitable medium and harvesting at least onehuman recombinant protein from the cell and/or the medium. Until theinvention, few, if any, human cells exist that have been found suitableto produce human recombinant proteins in any reproducible andupscaleable manner. We have now found that cells which include at leastimmortalizing adenoviral E1 sequences in their genome are capable ofgrowing (they are immortalized by the presence of E1) relativelyindependent of exogenous growth factors. Furthermore, these cells arecapable of producing recombinant proteins in significant amounts and arecapable of correctly processing the recombinant protein being made. Ofcourse, these cells will also be capable of producing non-humanproteins. The human cell lines that have been used to producerecombinant proteins in any significant amount are often tumor(transformed) cell lines. The fact that most human cells that have beenused for recombinant protein production are tumor-derived adds an extrarisk to working with these particular cell lines and results in verystringent isolation procedures for the recombinant protein in order toavoid transforming activity or tumorigenic material in any protein orother preparations. According to the invention, it is, therefore,preferred to employ a method wherein the cell is derived from a primarycell. In order to be able to grow indefinitely, a primary cell needs tobe immortalized in some kind, which, in the invention, has been achievedby the introduction of adenovirus E1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic drawing of the invention. In a first step (A), arecombinant adenoviral vector with deletions in E1 and E2A is propagatedin a first type of complementing cells that express both E1 and E2A ofadenovirus (here PER.E2A cells) (the recombinant adenoviral vector inthis Fig. is generated in situ by recombination of a plasmid containingthe left end of the adenovirus genome and a transgene encoding theprotein of interest with a cosmid containing the remainder of theadenovirus genome, inside the complementing cell, but other methods areequally suitable). In this step, recombinant adenovirus particles areformed (ΔE1/ΔE2A vector). In a second step (B), the recombinantadenovirus particles are used to infect a second type of complementingcells that express E1, but not E2A (here PER.C6 cells), so that theadenovirus particles can infect and produce protein in these cells, butcannot replicate and form adenovirus progeny in these cells. Thisresults in very efficient (high yield, fast) transient production of theprotein of interest.

FIG. 2. Analysis of S565 generated in PER.C6 cells, 3 days afterinfection with various MOI's of Ad5.dE1dE2A.S565 viruses. PER.C6 cellcultures were infected with 0, 100, 250, 500, 1000, 1500 and 2000vp/cell Ad5.dE1.dE2A.S565 (lanes 1-7). Medium samples were separatedusing SDS-PAGE and analyzed on western blot. S565 was detected usinganti-c-myc monoclonal antibody. Molecular weight marker (M) is depictedin the picture. Purified S565 transiently produced by plasmidtransfection in HEK293 cells was included as positive control (+). Seeexample 36 for details.

FIG. 3. Virus mediated SARS-AB production in PER.C6 cells afterinfection with various MOI's of Ad5.dE1.dE2A.SARS-AB viruses. Fivesimilar PER.C6 cultures (shaker flasks) were infected in parallel with0, 30, 60, 120 or 240 vp/cell Ad5.dE1.dE2A.SARS-AB. SARS-ABconcentration in medium samples was determined using HPLC-protA columnchromatography standardized for IgG quantity measurement. See example 36for details.

FIG. 4. Integrity of transiently expressed and purified S565, s.ACE2 andSARS-AB proteins. Roller bottle PER.C6 cultures were infected with 30vp/cell of Ad5.dE1dE2A.S565, Ad5.dE1dE2A.s.ACE2 or Ad5.dE1.dE2A.SARS-ABviruses. Proteins were separated on SDS-PAGE gels and analyzed onwestern blots. S565 and s.ACE2 were visualized with mouse-anti-c.myc(HRP-conjugated) monoclonal antibody. SARS-AB was visualized usingdonkey-anti-human IgG (HRP-conjugated) monoclonal antibody using anECL-detection system. Molecular weight marker (M) is depicted in thepicture. Purified S565 transiently produced in HEK293 cells (P), andstably produced CR3014 (anti-SARS antibody) on PER.C6 cells wereincluded as positive controls (C). See example 36 for details.

FIG. 5. Virus-mediated SARS-AB production in PER.C6 culture medium afterinfection with either purified or crude lysate preparations ofAd5.dE1.dE2A.SARS-AB viruses. Two similar cell cultures were infected inparallel with 1×10⁹ infectious virus particles (4 IU/cell) of eitherCsCl-purified vector (triangles) or with crude lysates containing thevector (squares). See example 36 for details.

FIG. 6. SDS-PAGE analysis, under non-reduced conditions, of purifiedSARS-AB batches transiently produced in PER.C6 cells. Cell cultures wereinfected in parallel with 4 IU/cell of purified virus (P) and crudelysates (C) of Ad5.dE1.dE2A.SARS-AB. Molecular weight marker (M) isindicated. Stably produced SARS-AB was included as positive control (+).See example 36 for details.

FIG. 7. A) Binding of CR3014 (anti-SARS antibody produced in stablytransfected PER.C6 cells) to transiently produced S565.

B) Binding of SARS-AB to S565. Two different SARS-AB batches (SARS-AB-1and SARS-AB-2), both produced in PERC.6 cells, after infection withpurified virus and crude lysate respectively, were tested and comparedto CR3014, a positive control SARS-AB. Amount of SARS-AB bound to S565was detected after staining with by FACS. See example 37 for details.

FIG. 8. FACS analysis demonstrating the ability of S1-spike to bindSARS-AB. S1-spike was transiently expressed in PER.C6 cells afterinfection with 30 and 100 vp/cell of Ad5.dE1.dE2A.S1-spike. Binding oftransiently produced SARS-AB was analyzed by FACS aftermouse-anti-c-myc-FITC Mab binding. Infected PER.C6 cells (both MOI's)stained only with mouse-anti-c-myc-FITC (no SARS-AB) are depicted asnegative controls. See example 37 for details.

FIG. 9. FACS analysis demonstrating s.ACE2 binding to S1-spike. S1-spikewas transiently expressed in PER.C6 cells using Ad5.dE1.dE2A.S1-spike.FACS staining was performed using transiently produced s.ACE2 (40 μg/ml)and subsequently stained with mouse-anti-c-myc-FITC Mab. Infected PER.C6cells stained only with mouse-anti-c-myc-FITC are depicted as negativecontrols. See example 37 for details.

FIG. 10. Analysis of transiently expressed ACE2 on cell membranes ofPER.C6 cells. Shaker flasks and roller bottles were infected in parallelwith MOI 0, 30, 60 and 120 vp/cell Ad5.dE1.dE2A.ACE2 viruses. For FACSanalysis cells were stained with anti-SARSspike IgG and PE-conjugatedanti-human IgG (100-fold dilution). Uninfected PER.C6 similarly stainedwere used as negative control. See example 37 for details.

DETAILED DESCRIPTION

In order to achieve large-scale (continuous) production of recombinantproteins through cell culture, it is preferred to have cells capable ofgrowing without the necessity of anchorage. The cells of the inventionhave that capability. The anchorage-independent growth capability isimproved when the cells include a sequence encoding E2A or a functionalderivative or analogue or fragment thereof in its genome, whereinpreferably the E2A encoding sequence encodes a temperature sensitivemutant E2A, such as ts125. To have a clean, relatively safe productionsystem from which it is easy to isolate the desired recombinant protein,it is preferred to have a method according to the invention, wherein thehuman cell includes no other adenoviral sequences. The most preferredcell for the methods and uses of the invention is PER.C6 as depositedunder ECACC no. 96022940 or a derivative thereof (see, e.g., U.S. Pat.No. 5,994,128 to Fallaux et al. (Nov. 30, 1999), the contents of whichare incorporated by this reference). PER.C6 cells behave better inhandling than, for instance, transformed human 293 cells that have alsobeen immortalized by the E1 region from adenovirus (Graham et al.,1977). PER.C6 cells have been characterized and have been documentedvery extensively because they behave significantly better in the processof up-scaling, suspension growth and growth factor independence.Especially the fact that PER.C6 cells can be brought in suspension in ahighly reproducible manner is something that makes it very suitable forlarge-scale production. Furthermore, the PER.C6 cell line has beencharacterized for bioreactor growth in which it grows to very highdensities.

The cells according to the invention, in particular PER.C6 cells, havethe additional advantage that they can be cultured in the absence ofanimal- or human-derived serum or animal- or human-derived serumcomponents. Thus isolation is easier, while the safety is enhanced dueto the absence of additional human or animal proteins in the culture,and the system is very reliable (synthetic media are the best inreproducibility). Furthermore, the presence of the Early region 1A(“E1A”) of adenovirus adds another level of advantages as compared to(human) cell lines that lack this particular gene. E1A as atranscriptional activator is known to enhance transcription from theenhancer/promoter of the CMV Immediate Early genes (Olive et al., 1990,Gorman et al., 1989). When the recombinant protein to be produced isunder the control of the CMV enhancer/promoter, expression levelsincrease in the cells and not in cells that lack E1A.

Further provided is a method for enhancing production of a recombinantproteinaceous substance in a eukaryotic cell, including providing theeukaryotic cell with a nucleic acid encoding at least part of theproteinaceous substance, wherein the coding sequence is under control ofa CMV-promoter, an E1A promoter or a functional homologue, derivativeand/or fragment of either and further providing the cell with E1Aactivity or E1A-like activity. Like the CMV promoter, E1A promoters aremore active in cells expressing one or more E1A products than in cellsnot expressing such products. It is known that indeed the E1A expressionenhancement is a characteristic of several other promoters. For suchmethods, such promoters are considered to be functional homologues of E1A promoters. The E1A effect can be mediated through the attraction oftranscription activators, the E1A promoter or homologue thereof, and/orthrough the removal/avoiding attachment of transcriptional repressors tothe promoter. The binding of activators and repressors to a promoteroccurs in a sequence-dependent fashion. A functional derivative and/orfragment of an E1A promoter or homologue thereof, therefore, at leastincludes the nucleic acid binding sequence of at least one E1A proteinregulated activator and/or repressor.

Another advantage of such cells is that they harbor and expressconstitutively the adenovirus E1B gene. Adenovirus E1B is a well-knowninhibitor of programmed cell death, or apoptosis. This inhibition occurseither through the 55K E1B product by its binding to the transcriptionfactor p53 or subsequent inhibition (Yew and Berk 1992). The otherproduct of the E1B region, 19K E1B, can prevent apoptosis by binding andthereby inhibiting the cellular death proteins Bax and Bak, bothproteins that are under the control of p53 (White et al., 1992; Debbasand White, 1993; Han et al., 1996; and Farrow et al., 1995). Thesefeatures can be extremely useful for the expression of recombinantproteins that, when over-expressed, might be involved in the inductionof apoptosis through a p53-dependent pathway.

Further provided is the use of a human cell for the production of ahuman recombinant protein, the cell having a sequence encoding at leastan immortalizing E1 protein of an adenovirus or a functional derivative,homologue or fragment thereof in its genome, which cell does not producestructural adenoviral proteins. In certain embodiments, the inventionprovides such a use wherein the human cell is derived from a primarycell, preferably wherein the human cell is a PER.C6 cell or a derivativethereof.

Further provided is a use according to the invention, wherein the cellfurther includes a sequence encoding E2A or a functional derivative oranalogue or fragment thereof in its genome, preferably wherein the E2Ais temperature sensitive.

Also provided is a human recombinant protein obtainable by a method orby a use disclosed herein, the human recombinant protein having a humanglycosylation pattern different from the isolated natural humancounterpart protein.

In certain embodiments, the invention provides a human cell having asequence encoding E1 of an adenovirus or a functional derivative,homologue or fragment thereof in its genome, which cell does not producestructural adenoviral proteins, and having a gene encoding a humanrecombinant protein, preferably a human cell which is derived fromPER.C6 as deposited under ECACC no. 96022940.

In yet another embodiment, the invention provides such a human cell,PER.C6/E2A, which further includes a sequence encoding E2A or afunctional derivative or analogue or fragment thereof in its genome,preferably wherein the E2A is temperature sensitive.

The proteins to be expressed in these cells using the methods of theinvention are well known to persons skilled in the art. They arepreferably human proteins that undergo some kind of processing innature, such as secretion, chaperoned folding and/or transport,co-synthesis with other subunits, glycosylation, or phosphorylation.Typical examples for therapeutic or diagnostic use include monoclonalantibodies that are comprised of several subunits, tissue-specificplasminogen activator (“tPA”), granulocyte colony stimulating factor(“G-CSF”) and human erythropoietin (“EPO” or “hEPO”). EPO is a typicalproduct that, especially in vivo, heavily depends on its glycosylationpattern for its activity and immunogenicity. Thus far, relatively highlevels of EPO have been reached by the use of CHO cells which aredifferently glycosylated in comparison to EPO purified from human urine,albeit equally active in the enhancement of erythrocyte production. Thedifferent glycosylation of such EPO, however, can lead to immunogenicityproblems and altered half-life in a recipient.

The invention also includes a novel human immortalized cell line forthis purpose and the uses thereof for production. PER.C6 cells (PCTInternational Patent Publication WO 97/00326 or U.S. Pat. No. 5,994,128)were generated by transfection of primary human embryonic retina cellsusing a plasmid that contained the adenovirus serotype 5 (Ad5) E1A- andE1B-coding sequences (Ad5 nucleotides 459-3510) under the control of thehuman phosphoglycerate kinase (“PGK”) promoter.

The following features make PER.C6 particularly useful as a host forrecombinant protein production: (1) fully characterized human cell line;(2) developed in compliance with GRP; (3) can be grown as suspensioncultures in defined serum-free medium devoid of any human- oranimal-derived proteins; (4) growth compatible with roller bottles,shaker flasks, spinner flasks and bioreactors with doubling times ofabout 35 hours; (5) presence of E1A causing an up-regulation ofexpression of genes that are under the control of the CMVenhancer/promoter; (6) presence of E1B which prevents p53-dependentapoptosis possibly enhanced through overexpression of the recombinanttransgene.

In certain embodiments, the invention provides a method wherein the cellis capable of producing two- to 200-fold more recombinant protein and/orproteinaceous substance than conventional mammalian cell lines.Preferably, the conventional mammalian cell lines are selected from thegroup consisting of CHO, COS, Vero, Hela, BHK and Sp-2/0 cell lines.

Thus, it would be an improvement in the art to provide a human cell thatproduces consistent human-type protein processing likepost-translational and peri-translational modifications, such as, butnot limited to glycosylation. It would be further advantageous toprovide a method for producing a recombinant mammalian cell and proteinsfrom recombinant mammalian cells in large-scale production.

Previously, few, if any, human cells suitable for producing proteins inany reproducible and upscaleable manner have been found. The cells ofthe invention include at least an immortalizing adenoviral E1 proteinand are capable of growing relatively independent of exogenous growthfactors.

Furthermore, these cells are capable of producing proteins insignificant amounts and are capable of correctly processing thegenerated immunoglobulins.

The fact that cell types that have been used for protein production aretumor-derived adds an extra risk to working with these particular celllines and results in very stringent isolation procedures for theproteins in order to avoid transforming activity or tumorigenic materialin any preparations. It is, therefore, preferred to employ a methodaccording to the invention, wherein the cell is derived from a primarycell. In order to be able to grow indefinitely, a primary cell needs tobe immortalized, which in the invention has been achieved by theintroduction of an adenoviral E1 protein.

In order to achieve large-scale (continuous) production of proteinsthrough cell culture, it is preferred to have cells capable of growingwithout the necessity of anchorage. The cells of the invention have thatcapability. The anchorage-independent growth capability is improved whenthe cells include an adenovirus-derived sequence encoding E2A (or afunctional derivative or analogue or fragment thereof) in its genome. Ina preferred embodiment, the E2A encoding sequence encodes a temperaturesensitive mutant E2A, such as ts125. The cell may, in addition, includea nucleic acid (e.g., encoding tTa), which allows for regulatedexpression of a gene of interest when placed under the control of apromoter (e.g., a TetO promoter).

To have a clean and safe production system from which it is easy toisolate the desired proteins, it is preferred to have a method accordingto the invention, wherein the human cell includes no other adenoviralsequences. The most preferred cell for the methods and uses of theinvention is a PER.C6 cell (or a derivative thereof) as deposited underECACC no. 96022940. PER.C6 cells have been found to be more stable,particularly in handling, than, for instance, transformed human 293cells immortalized by the adenoviral E1 region. PER.C6 cells have beenextensively characterized and documented, demonstrating good process ofupscaling, suspension growth and growth factor independence.Furthermore, PER.C6 can be incorporated into a suspension in a highlyreproducible manner, making it particularly suitable for large-scaleproduction. In this regard, the PER.C6 cell line has been characterizedfor bioreactor growth, where it can grow to very high densities.

The cells of the invention, in particular PER.C6, can advantageously becultured in the absence of animal- or human-derived serum, or animal- orhuman-derived serum components. Thus, isolation of proteins issimplified and safety is enhanced due to the absence of additional humanor animal proteins in the culture. The absence of serum furtherincreases reliability of the system since use of synthetic media, ascontemplated herein, enhances reproducibility.

Further provided is the use of a recombinant mammalian cell for theproduction of at least one polypeptide, the cell having a sequenceencoding at least an immortalizing E1 protein of an adenovirus or afunctional derivative, homologue or fragment thereof in its genome,which cell does not produce structural adenoviral proteins. In certainembodiments, the invention provides such a use wherein the cell isderived from a primary cell, preferably wherein the human cell is aPER.C6 cell or a derivative thereof.

Further provided is a use according to the invention, wherein the cellfurther includes a sequence encoding E2A (or a functional derivative oranalogue or fragment thereof) in its genome, preferably wherein the E2Ais temperature sensitive. In addition, the invention provides a methodof using the invention, wherein the cell further includes atrans-activating protein for the induction of the inducible promoter.Also provided is proteins obtainable by a method according to theinvention or by a use according to the invention.

In certain embodiments, the invention provides a human cell having asequence encoding E1 of an adenovirus (or a functional derivative,homologue or fragment thereof) in its genome, which cell does notproduce structural adenoviral proteins, and having a gene encoding ahuman recombinant protein, preferably a human cell which is derived fromPER.C6 as deposited under ECACC No. 96022940.

In yet another embodiment, the invention provides such a human cell,PER.C6/E2A, which further includes a sequence encoding E2A (or afunctional derivative, analogue or fragment thereof) in its genome,preferably wherein the E2A is temperature sensitive.

Further provided is methods for producing at least one polypeptide in arecombinant mammalian cell utilizing the immortalized recombinantmammalian cell of the invention, culturing the same in a suitablemedium, and harvesting at least one polypeptide from the recombinantmammalian cell and/or medium. The recombinant polypeptides, orderivatives thereof, may be used for the therapeutic treatment ofmammals or the manufacture of pharmaceutical compositions.

In certain embodiments, a cell is derived from a human primary cell,preferably a cell which is immortalized by a gene product of the E1gene. In order to be able to grow, a primary cell, of course, needs tobe immortalized. A good example of such a cell is one derived from ahuman embryonic retinoblast.

In cells according to the invention, it is important that the E1 genesequences are not lost during the cell cycle. It is, therefore,preferred that the sequence encoding at least one gene product of the E1gene is present in the genome of the (human) cell. For reasons ofsafety, care is best taken to avoid unnecessary adenoviral sequences inthe cells according to the invention. It is thus another embodiment ofthe invention to provide cells that do not produce adenoviral structuralproteins. However, in order to achieve large-scale (continuous) virusprotein production through cell culture, it is preferred to have cellscapable of growing without needing anchorage. The cells of the inventionhave that capability. To have a clean and safe production system fromwhich it is easy to recover and, if desirable, to purify the recombinantprotein, it is preferred to have a method according to the invention,wherein the human cell includes no other adenoviral sequences. The mostpreferred cell for the methods and uses of the invention is PER.C6 asdeposited under ECACC no. 96022940, or a derivative thereof.

Thus, provided is a method using a cell according to the invention,wherein the cell further includes a sequence encoding E2A or afunctional derivative or analogue or fragment thereof, preferably a cellwherein the sequence encoding E2A or a functional derivative or analogueor fragment thereof is present in the genome of the human cell, and mostpreferably a cell wherein the E2A encoding sequence encodes atemperature sensitive mutant E2A.

Furthermore, as stated, also provided is a method according to theinvention wherein the (human) cell is capable of growing in suspension.

Also described is a method wherein the human cell can be cultured in theabsence of serum. The cells according to the invention, in particularPER.C6® cells, have the additional advantage that they can be culturedin the absence of serum or serum components. Thus, isolation is easy,safety is enhanced and reliability of the system is good (syntheticmedia are the best in reproducibility). The human cells of theinvention, and in particular those based on primary cells andparticularly the ones based on HER cells, are capable of normal post andperi-translational modifications and assembly. This means that they arevery suitable for preparing proteins for use in therapeuticapplications.

Thus, the invention also includes a method wherein the protein includesa protein that undergoes post-translational and/or peri-translationalmodification, especially wherein the modifications includeglycosylation.

In another aspect, the invention provides the use of an adenoviral E1Bprotein or a functional derivative, homologue and/or fragment thereofhaving anti-apoptotic activity for enhancing the production of aproteinaceous substance in a eukaryotic cell, the use includingproviding the eukaryotic cell with the E1B protein, derivative,homologue and/or fragment thereof. In a preferred embodiment, the useincludes a cell of the invention. In certain embodiments, the inventionprovides such a use in a method and/or a use described herein.

In certain embodiments, the proteinaceous substance or protein is amonoclonal antibody. Antibodies, or immunoglobulins (“Igs”), are serumproteins that play a central role in the humoral immune response,binding antigens and inactivating them or triggering the inflammatoryresponse which results in their elimination. Antibodies are capable ofhighly specific interactions with a wide variety of ligands, includingtumor-associated markers, viral coat proteins, and lymphocyte cellsurface glycoproteins. They are, therefore, potentially very usefulagents for the diagnosis and treatment of human diseases. Recombinantmonoclonal and single chain antibody technology is opening newperspectives for the development of novel therapeutic and diagnosticagents. Mouse monoclonal antibodies have been used as therapeutic agentsin a wide variety of clinical trials to treat infectious diseases andcancer. The first report of a patient being treated with a murinemonoclonal antibody was published in 1980 (Nadler et al. 1980). However,the effects observed with these agents have, in general, been quitedisappointing (for reviews, see Lowder et al. 1985, Mellstedt et al.1991, Baldwin and Byers 1985). Traditionally, recombinant monoclonalantibodies (immunoglobulins) are produced on B-cell hybridomas. Suchhybridomas are produced by fusing an immunoglobulin-producing B-cell,initially selected for its specificity, to a mouse myeloma cell andthereby immortalizing the B-cell. The original strategy of immortalizingmouse B-cells was developed in 1975 (Köhler and Milstein). However,immunoglobulins produced in such hybridomas have the disadvantage thatthey are of mouse origin, resulting in poor antibody specificity, lowantibody affinity and a severe host anti-mouse antibody response (HAMA,Shawler et al. 1985). This HAMA response may lead to inflammation,fever, and even death of the patient.

Mouse antibodies have a low affinity in humans and, for reasons yetunknown, have an extremely short half-life in human circulation (19-42hours) as compared to human antibodies (21 days, Frödin et al., 1990).That, together with the severity of the HAMA response, has prompted thedevelopment of alternative strategies for generating more human orcompletely humanized immunoglobulins (reviewed by Owens and Young 1994,Sandhu 1992, Vaswani et al. 1998).

One such strategy makes use of the constant regions of the humanimmunoglobulin to replace its murine counterparts, resulting in a newgeneration of “chimeric” and “humanized” antibodies. This approach istaken since the HAMA response is mainly due to the constant domains (Oiet al., 1983; Morrison et al., 1984). An example of such a chimericantibody is CAMPATH-1H (Reichmann et al. 1988). The CAMPATH-1H Ab, usedin the treatment of non-Hodgkin's B-cell lymphoma and refractoryrheumatoid arthritis, is directed against the human antigen CAMPATH-1(CDw52) present on all lymphoid cells and monocytes but not on othercell types (Hale et al. 1988, Isaacs et al. 1992). Other examples areRituxan (Rituximab) directed against human CD20 (Reff et al. 1994) and15C5, a chimeric antibody raised against human fragment-D dimer(Vandamme et al. 1990, Bulens et al. 1991) used in imaging of bloodclotting. However, since these new generation chimeric antibodies arestill partly murine, they can induce an immune response in humans,albeit not as severe as the HAMA response against fully murineantibodies of mouse origin.

In another, more sophisticated approach, ranges of residues present inthe variable domains of the antibody, but apparently not essential forantigen recognition, are replaced by more human-like stretches of aminoacids, resulting in a second generation or hyperchimeric antibodies(Vaswani et al. 1998). A well-known example of this approach isHerceptin (Carter et al. 1992), an antibody that is 95% human, which isdirected against HER2 (a tumor-specific antigen) and used in breasttumor patients.

A more preferred manner to replace mouse recombinant immunoglobulinswould be one resulting in the generation of human immunoglobulins.Importantly, since it is unethical to immunize humans with experimentalbiological materials, it is not feasible to subsequently select specificB-cells for immortalization as was shown for mouse B-cells (Köhler andMilstein 1975). Although B-cells from patients were selected forspecific antibodies against cancer antigens, it is technically moredifficult to prepare human immunoglobulins from human material ascompared to mouse antibodies (Köhler and Milstein, 1975). A recombinantapproach to produce fully human antibodies became feasible with the useof phage displayed antibody libraries, expressing variable domains ofhuman origin (McCafferty et al. 1990, Clarkson et al. 1991, Barbas etal. 1991, Garrard et al. 1991, Winter et al. 1994, Burton and Barbas,1994). These variable regions are selected for their specific affinityfor certain antigens and are subsequently linked to the constant domainsof human immunoglobulins, resulting in human recombinantimmunoglobulins. An example of this latter approach is the single chainFv antibody 17-1A (Riethmuller et al. 1994) that was converted into anintact human IgG1 kappa immunoglobulin named UBS-54, directed againstthe tumor-associated EpCAM molecule (Huls et al. 1999).

The production systems to generate recombinant immunoglobulins arediverse. The mouse immunoglobulins first used in clinical trials wereproduced in large quantities in their parental-specific B-cell and fusedto a mouse myeloma cell for immortalization. A disadvantage of thissystem is that the immunoglobulins produced are entirely of mouse originand render a dramatic immune response (HAMA response) in the humanpatient (as previously described herein).

Partially humanized or human antibodies lack a parental B-cell that canbe immortalized and therefore have to be produced in other systems likeCHO cells or Baby Hamster Kidney (BHK) cells. It is also possible to usecells that are normally suited for immunoglobulin production liketumor-derived human or mouse myeloma cells. However, antibody yieldsobtained in myeloma cells are, in general, relatively low (±0.1 ug/ml)when compared to those obtained in the originally identified andimmortalized B-cells that produce fully murine immunoglobulins (±10ug/ml, Sandhu 1992).

To circumvent these and other shortcomings, different systems are beingdeveloped to produce humanized or human immunoglobulins with higheryields.

For example, it was recently shown that transgenic mouse strains can beproduced that have the mouse IgG genes replaced with their humancounterparts (Bruggeman et al., 1991, Lonberg et al., 1994, Lonberg andHuszar, 1995, Jacobovits, 1995). Yeast artificial chromosomes (“YACs”)containing large fragments of the human heavy and light (kappa) chainimmunoglobulin (Ig) loci were introduced into Ig-inactivated mice,resulting in human antibody production which closely resembled that seenin humans, including gene rearrangement, assembly, and repertoire(Mendez et al. 1997, Green et al. 1994). Likewise, Fishwild et al.(1996) have constructed human Ig-transgenics in order to obtain humanimmunoglobulins using subsequent conventional hybridoma technology. Thehybridoma cells secreted human immunoglobulins with properties similarto those of wild-type mice including stability, growth, and secretionlevels. Recombinant antibodies produced from such transgenic micestrains carry no non-human amino acid sequences.

Nevertheless, human immunoglobulins produced thus far have thedisadvantage of being produced in non-human cells, resulting innon-human post-translational modifications like glycosylation and/orfolding of the subunits. All antibodies are glycosylated at conservedpositions in their constant regions, and the presence of carbohydratescan be critical for antigen clearance functions such as complementactivation. The structure of the attached carbohydrate can also affectantibody activity. Antibody glycosylation can be influenced by the cellin which it is produced, the conformation of the antibody and cellculture conditions. For instance, antibodies produced in mouse cellscarry glycans containing the Gal alpha1-3Gal residue, which is absent inproteins produced in human cells (Borrebaeck et al. 1993, Borrebaeck,1999). A very high titer of anti-Gal alpha1-3Gal antibodies is presentin humans (100 ug/ml, Galili, 1993), causing a rapid clearance of(murine) proteins carrying this residue in their glycans.

It soon became apparent that, in order to exert an effect, patients needto be treated with very high doses of recombinant immunoglobulins forprolonged periods of time. It seems likely that post-translationalmodifications on human or humanized immunoglobulins that are notproduced on human cells strongly affect the clearance rate of theseantibodies from the bloodstream.

It is unclear why immunoglobulins produced on CHO cells also need to beapplied in very high dosages, since the Gal alpha1-3Gal residue is notpresent in glycans on proteins derived from this cell line (Rother andSquinto, 1996). Therefore, other post-translational modificationsbesides the Gal alpha1-3Gal residues are likely to be involved inspecific immune responses in humans against fully human or humanizedimmunoglobulins produced on such CHO cells.

The art thus teaches that it is possible to produce humanized antibodieswithout murine-derived protein sequences. However, the currentgeneration of recombinant immunoglobulins still differs from its naturalhuman counterparts, for example, by post-translational modificationssuch as glycosylation and folding. This may result in activation of theimmune system of the patient and cause undesirable responses that mayaffect the efficacy of the treatment. Thus, despite the development ofchimeric antibodies, the current production systems still needoptimization to produce fully human or humanized active antibodies.

It is thus clearly desirable to have methods for producing fully humanantibodies which behave accordingly, and which are, in addition,produced at higher yields than observed in human myeloma cells.

Thus, it would be an improvement in the art to provide a human cell thatproduces consistent human-type protein processing likepost-translational and peri-translational modifications, such as, butnot limited to glycosylation. It would be further advantageous toprovide a method for producing a recombinant mammalian cell andimmunoglobulins from recombinant mammalian cells in large-scaleproduction.

Therefore, further provided is a method for producing at least onevariable domain of an immunoglobulin in a recombinant mammalian cell,including providing a mammalian cell including a nucleic acid encodingat least an immortalizing E1 protein of an adenovirus or a functionalderivative, homologue and/or fragment thereof in its genome, and furtherincluding a second nucleic acid encoding the immunoglobulin, culturingthe cell in a suitable medium and harvesting at least one monoclonalantibody from the cell and/or the medium.

Previously, few, if any, human cells suitable for producingimmunoglobulins in any reproducible and upscaleable manner have beenfound. The cells of the invention include at least an immortalizingadenoviral E1 protein and are capable of growing relatively independentof exogenous growth factors.

Furthermore, these cells are capable of producing immunoglobulins insignificant amounts and are capable of correctly processing thegenerated immunoglobulins.

The fact that cell types that have been used for immunoglobulinproduction are tumor-derived adds an extra risk to working with theseparticular cell lines and results in very stringent isolation proceduresfor the immunoglobulins in order to avoid transforming activity ortumorigenic material in any preparations. It is therefore preferred toemploy a method according to the invention, wherein the cell is derivedfrom a primary cell. In order to be able to grow indefinitely, a primarycell needs to be immortalized, which in the invention has been achievedby the introduction of an adenoviral E1 protein.

In order to achieve large-scale (continuous) production ofimmunoglobulins through cell culture, it is preferred to have cellscapable of growing without the necessity of anchorage. The cells of theinvention have that capability. The anchorage-independent growthcapability is improved when the cells include an adenovirus-derivedsequence encoding E2A (or a functional derivative or analogue orfragment thereof) in its genome. In a preferred embodiment, the E2Aencoding sequence encodes a temperature sensitive mutant E2A, such asts125. The cell may, in addition, include a nucleic acid (e.g., encodingtTa), which allows for regulated expression of a gene of interest whenplaced under the control of a promoter (e.g., a TetO promoter).

The nucleic acid may encode a heavy chain, a variable heavy chain, alight chain, and/or a variable light chain of an immunoglobulin.Alternatively, a separate or distinct nucleic acid may encode one ormore variable domain(s) of an Ig (or a functional derivative, homologueand/or fragment thereof) as a counterpart to the first nucleic acid(described above). One or more nucleic acid(s) described herein mayencode an ScFv and may be human or humanized. The nucleic acid(s) of theinvention can in certain embodiments be placed under the control of aninducible promoter (or a functional derivative thereof).

To have a clean and safe production system from which it is easy toisolate the desired immunoglobulins, it is preferred to have a methodaccording to the invention, wherein the human cell includes no otheradenoviral sequences in its genome. The most preferred cell for themethods and uses of the invention is PER.C6 or a derivative thereof asdeposited under ECACC no. 96022940. PER.C6 has been found to be morestable, particularly in handling, than, for instance, transformed human293 cells immortalized by the adenoviral E1 region. PER.C6 cells havebeen extensively characterized and documented, demonstrating goodprocess of upscaling, suspension growth and growth factor independence.Furthermore, PER.C6 can be incorporated into a suspension in a highlyreproducible manner, making it particularly suitable for large-scaleproduction. In this regard, the PER.C6 cell line has been characterizedfor bioreactor growth, where it can grow to very high densities.

The cells of the invention, in particular PER.C6, can advantageously becultured in the absence of animal- or human-derived serum, or animal- orhuman-derived serum components. Thus, isolation of monoclonal antibodiesis simplified and safety is enhanced due to the absence of additionalhuman or animal proteins in the culture. The absence of serum furtherincreases reliability of the system since use of synthetic media, ascontemplated herein, enhances reproducibility.

Further provided is the use of a recombinant mammalian cell for theproduction of at least one variable domain of an immunoglobulin, thecell having a sequence encoding at least an immortalizing E1 protein ofan adenovirus or a functional derivative, homologue or fragment thereofin its genome, which cell does not produce structural adenoviralproteins. In certain embodiments, the invention provides such a usewherein the cell is derived from a primary cell, preferably wherein thehuman cell is a PER.C6 cell or a derivative thereof.

Further provided is a use, wherein the cell further includes a sequenceencoding E2A (or a functional derivative or analogue or fragmentthereof) in its genome, preferably wherein the E2A is temperaturesensitive. In addition, the invention provides a method of using theinvention, wherein the cell further includes a trans-activating proteinfor the induction of the inducible promoter. Also provided isimmunoglobulins obtainable by a method according to the invention or bya use according to the invention.

Immunoglobulins to be expressed in the cells of the invention are knownto persons skilled in the art. Examples of recombinant immunoglobulinsinclude, but are not limited to, Herceptin®, Rituxan (Rituximab®),UBS-54, CAMPATH-1H®, and 15C5.

Further provided are methods for producing at least one variable domainof an immunoglobulin in a recombinant mammalian cell utilizing theimmortalized recombinant mammalian cell of the invention, culturing thesame in a suitable medium, and harvesting at least one variable domainof a selected Ig from the recombinant mammalian cell and/or medium.Immunoglobulins, variable domains of the immunoglobulins, or derivativesthereof may be used for the therapeutic treatment of mammals or themanufacture of pharmaceutical compositions.

In certain embodiments, further provided is a method for producing aviral protein other than adenovirus or adenoviral protein for use as avaccine including providing a cell with at least a sequence encoding atleast one gene product of the E1 gene or a functional derivative thereofof an adenovirus, providing the cell with a nucleic acid encoding theviral protein, culturing the cell in a suitable medium allowing forexpression of the viral protein and harvesting viral protein from themedium and/or the cell. Until the invention, there are few, if any(human), cells that have been found suitable to produce viral proteinsfor use as vaccines in any reproducible and upscaleable manner and/orsufficiently high yields and/or easily purifiable. We have now foundthat cells which include adenoviral E1 sequences, preferably in theirgenome, are capable of producing the viral protein in significantamounts.

Thus, the invention also includes a method wherein the viral proteinincludes a protein that undergoes post-translational and/orperi-translational modification, especially wherein the modificationsinclude glycosylation. A good example of a viral vaccine that has beencumbersome to produce in any reliable manner is influenza vaccine. Theinvention provides a method according to the invention wherein the viralproteins include at least one of an influenza virus neuramidase and/or ahemagglutinin. Other viral proteins (subunits) that can be produced inthe methods according to the invention include proteins fromenterovirus, such as rhinovirus, aphto virus, or poliomyelitis virus,herpes virus, such as herpes simplex virus, pseudorabies virus or bovineherpes virus, orthomyxovirus, such as influenza virus, a paramyxovirus,such as Newcastle Disease virus, respiratory syncitio virus, mumps virusor a measles virus, retrovirus, such as human immunodeficiency virus ora parvovirus or a papova virus, rotavirus or a coronavirus, such astransmissible gastroenteritis virus or a flavivirus, such as tick-borneencephalitis virus or yellow fever virus, a togavirus, such as rubellavirus or eastern-, western-, or Venezuelan equine encephalomyelitisvirus, a hepatitis causing virus, such as hepatitis A or hepatitis Bvirus, a pestivirus, such as hog cholera virus or a rhabdovirus, such asrabies virus.

Also provided is the products resulting from the methods and usesaccording to the invention, especially viral proteins obtainableaccording to those uses and/or methods, especially when brought in apharmaceutical composition including suitable excipients and in someformats (subunits) adjuvants. Dosage and ways of administration can besorted out through normal clinical testing if they are not yet availablethrough the already registered vaccines.

Thus, also provided is a viral protein for use in a vaccine obtainableby a method or by a use according to the invention, the viral proteinbeing free of any non-human mammalian proteinaceous material and apharmaceutical formulation including such a viral protein.

In a preferred embodiment, the invention provides influenza vaccinesobtainable by a method according to the invention or by a use accordingto the invention.

As shown in U.S. Pat. No. 6,855,544 (the '544 patent) to Hateboer et al,the contents of the entirety of which are incorporated by thisreference, immortalized human embryonic retina cells expressing at leastan adenovirus E1A protein, such as PER.C6 cells, can be suitably usedfor the production of recombinant proteins. In preferred aspects in the'544 patent, the transgene encoding the protein of interest isintegrated into the cells genome. The advantage of such methods is thatlong-term stable expression can be obtained, because the transgene isstably inherited. This is particularly advantageous when recombinantproteins are to be used in pharmaceutical applications, where stabilityand robustness is of paramount importance, for instance to satisfy thestrict regulatory requirements imposed by governing authorities.Moreover, large quantities of recombinant protein can be obtained once asuitable cell line has been selected.

However, a disadvantage of methods wherein stable clones are used, isthat it takes relatively long (at least several months) and relativelymuch hands-on time of laboratory personnel to obtain the stable clonewith the desired characteristics. Therefore, transient expressionmethods are seen in some circumstances as an alternative to stableexpression methods (e.g., Wurm and Bernard, 1999; Baldi et al, 2007), inparticular to obtain limited amounts of material that can be used forpre-clinical testing and in some cases even for phase I clinical trials.Efforts have been made to scale-up transient transfection methods insuspension for certain cell lines to 100 liter scale, so that sufficientmaterial for clinical trials can in principle be obtained. In transientexpression methods, a plasmid containing the transgene is transfectedinto the host cells, generally without applying selection, and thetransiently expressed protein is harvested from the pool of transfectedcells (i.e., without generating stable clones). As shown in theincorporated '544 patent, PER.C6 cells can also be suitably used fortransient expression of proteins. The advantage of transient expressionmethods is the limited time required to obtain some material of theprotein of interest (depending somewhat on the scale, but most effort isrequired to obtain sufficient cells and plasmid to be transfected, whilewithin a few days after transfection the desired protein can beharvested, so that the total time required for obtaining material ingeneral is not more than a few weeks). A disadvantage of transienttransfection with plasmids in suspension is its inherent inefficiency,because i) DNA is not easily taken up by cells, and ii) requiredtransport of the DNA from the cytoplasm where it is taken up into thenucleus where it is transcribed is inefficient. Therefore, only arelatively small subset of the cells of the transfected pool willcontribute to expression of the protein of interest (in transienttransfection with adherent cells efficient transfection is possible, butthose methods are not scalable to industrially useful amounts). Hence,expression levels in such transient suspension systems are usuallyorders of magnitude lower than those obtainable by recombinantexpression from stable cell clones. Furthermore, because of theseinherent disadvantages that become increasingly more apparent at largerscale, the scalability of these systems has its limits, and it isquestionable whether transient plasmid transfections for proteinproduction could become economically profitable beyond the 100 literscale. The best results reported thus far for a multiliter transfectionin CHO cells by highly specialized investigators in this field,described a recombinant antibody yield of 22 mg/l (Muller et al, 2007).

Disclose herein is a relatively clean and scalable transient expressionsystem for efficient high level expression of proteins of interest, byinfection of suitable cells with recombinant adenovirus vectors. Thissystem allows for clean ‘supertransfection’ of the cells, and has theadvantages of transient systems (fast), and does not, or to a much lessextent, have the disadvantages described above. For instance, becauseadenovirus vectors infect cells very efficiently and moreover delivertheir transgenes into the nucleus of the infected cell, the inherentdisadvantage described for the transient transfection with plasmids havebeen overcome, and using the system of the invention provides a veryhigh percentage of cultured cells that contribute to expression of theprotein. In addition, the system is very fast and thus has advantagesover the stable transfection methods described above. Moreover, thesystem is scalable, in principle to very large bioreactor scale (e.g.,10 1, 100 1, 500 1, 1000 1, 2000 1, 5000 1, 10000 1, 20000 1) since thehost cells can be cultured to very large scale according to knownmethods, and adenovirus biology will serve for very efficient infectionlevels and thus expression of the transgene in a very high proportion ofthe cells in the large culture. The system can thus be used atindustrial scale to prepare proteins of interest, for instance forcharacterization of the protein, pre-clinical testing, phase I testing,and in certain cases even for phase II testing. The system can also bedown-scaled to prepare small quantities of many different proteins fortesting some properties of a variety of candidate proteins, e.g., in amulti-well format. Our first results as reported herein for expressionof an antibody demonstrate a yield of 140 mg/l.

Recombinant adenoviruses are widely used in the field of gene therapyand in the field of vaccine preparation. In general in these fields, therecombinant adenovirus has a deletion in at least one region requiredfor replication of the adenovirus, e.g., the early region 1 (E1),providing space for a transgene of interest that can be expressed in thetarget cells. Because of the deletion of one essential region, theresulting virus is replication deficient in normal cells, and topropagate the virus a so-called complementing cell is used, thatprovides in trans the functions deleted from the adenovirus genome.Most, if not all, adenovirus vectors used today have a deletion in E1,and complementing cells providing the E1 function are used forcomplementation. Several such E1-complementing cells are known in theart, such as HEK293, 911, PER.C6, and many others. A particularly usefulcomplementing cell is a PER.C6 cell (U.S. Pat. No. 5,994,128,incorporated herein by reference). Construction of recombinantadenovirus and complementing cell lines that complement thecorresponding deficiencies in the genome of the recombinant adenovirushave been extensively described and is part of the knowledge of theskilled person, which can make such materials in a routine fashion basedon this knowledge, see e.g., U.S. Pat. Nos. 6,133,028; 5,994,128; and6,482,616, all incorporated in their entirety by reference herein.

The transient protein expression system disclosed herein makes use ofrecombinant adenovirus that has at least two deletions in the genome ofwild-type adenovirus. The deletions are in regions of the adenovirusgenome that are required for adenovirus genome replication and/or foradenovirus particle formation. The recombinant adenovirus genome furthercomprises a transgene, comprising nucleic acid encoding the protein ofinterest in expressible format. In preferred embodiments, the adenovirusgenome is deleted in the E1 region, which is thus one of the at leasttwo deletions. The other deletion is in a region of the adenovirusgenome that is required for adenovirus genome replication or adenovirusparticle formation, and in certain embodiments is in the E2A region. Ina first step, for propagation of the adenovirus, a complementation cellline is used that complements both deletions. For instance, for anadenovirus with deletions in E1 and E2A, a complementation cell thatexpresses both E1 and E2A is used in this step. In this manner,recombinant adenovirus particles are obtained, which contain a genomecomprising the transgene and having deletions in the two regions. Theseobtained adenovirus particles are used to infect a second type ofcomplementing cells, which complement only one of the two deletions. Inpreferred embodiments, the second type of complementing cells expressesadenoviral E1 protein. The recombinant adenovirus particles canefficiently infect these cells, but cannot be propagated therein or atleast not form infectious particles therein because the functionsrequired for adenovirus replication or particle formation are absent(i.e., both from the genome of the recombinant adenovirus and from thecomplementing cell). Therefore, these cells are infected with therecombinant adenovirus, but do not produce adenovirus particles unlikethe first complementing cell line. The infection however results in veryefficient transient production of the transgene of the adenovirus. Theprotein of interest is then harvested, without adenovirus particlesbeing produced. FIG. 1 shows a scheme of the method of the invention.

Others have used adenovirus vectors for production of recombinantproteins in complementing cell lines (Massie et al, 1995; Garnier et al,1994). However, those methods used adenovirus vectors with a deletion inE1 and infected E1-expressing complementing cells, so that adenovirusparticles could be formed. The instant invention has the advantage thatno adenovirus particles are formed in the final step during expressionof the transgene, and thus provides a cleaner system. This is veryimportant when proteins for pharmaceutical use are produced, since thepresence of adenovirus particles in the protein preparation will not beallowed in view of regulatory considerations, so that the methodsdescribed by others will require additional efforts to remove adenovirusparticles. Moreover, the cells do not have to spend energy in formingadenovirus particles, so that the cells are more committed to producingthe protein of interest, which may result in higher yields.

Multiply deficient adenovirus vectors, and cells for complementingthese, have previously been described by us and by others (e.g., U.S.Pat. Nos. 6,482,616, 6,395,519 and 6,133,028, incorporated herein bythis reference). However, in those cases, the cells were just used tocomplement the vectors, which were subsequently used for gene therapy orvaccine purposes, i.e., the generated recombinant adenoviral particleswere used to infect normal cells in a living organism. In contrast, theinvention comprises a step of infecting other cultured complementingcells for protein production. In the methods previously disclosed, thepurpose was never to produce recombinant protein in vitro in highquantities like in the current invention, but rather to infect cells invivo to obtain some expression therein in situ. The methods previouslydescribed are therefore further silent on harvesting the protein ofinterest (which was not the goal, nor possible), which is a step of thecurrently invented methods. Moreover, in the methods previouslydescribed, all deficiencies of the adenovirus vectors were complementedin the complementing cells, whereas methods of the invention include astep of infecting cells that do not complement all deficiencies in theadenovirus genome, and isolating protein after this step.

The invention thus provides a method for producing a protein ofinterest, the method comprising: a) providing a recombinant adenoviralvector comprising nucleic acid encoding the protein of interest undercontrol of a promoter, wherein the adenoviral vector has deletions in afirst region and in a second region of the adenovirus genome, whereineach of the first region and the second region is required foradenoviral genome replication and/or adenovirus particle formation, b)propagating the adenoviral vector in a first type of complementing cellsthat express proteins from the first and from the second region of theadenovirus genome so as to complement the deletions of the recombinantadenoviral vector, to obtain recombinant adenovirus particles, c)infecting a culture of a second type of complementing cells with therecombinant adenovirus particles, wherein the second type ofcomplementing cells express protein from the first region of theadenovirus genome but not protein from the second region of theadenovirus genome, to produce the protein of interest, and d) harvestingthe protein of interest.

In preferred embodiments, the first region of the adenovirus genome isthe E1 region. Nearly all adenovirus vectors that are currently usedhave a deletion in E1, and therefore several complementing cells thatexpress E1 protein are available. Any cell expressing E1 protein andsusceptible to adenovirus infection can be used. As non-limitingexamples, such cells can be derived from for instance human cells, forinstance from a kidney (example: HEK 293 cells, see Graham et al, 1977),lung (e.g., A549, see e.g., WO 98/39411) or retina (example: HER cellssuch as PER.C6 cells, see U.S. Pat. No. 5,994,128), or from amniocytes(e.g., N52.E6, described in U.S. Pat. No. 6,558,948), and similarly fromseveral other types of cells (see, e.g., Xu et al, Cytotechnology 51:133-140 (2006); DE 19754103, Kim J-S. et al. (2001) Experimental andMolecular Medicine 33: 145-149). Methods for obtaining such cells aredescribed for instance in U.S. Pat. Nos. 5,994,128 and 6,558,948. Thus,the second type of complementing cells, as used in step c) of theinvention, is readily available. Another advantage of embodimentswherein the first region of the adenovirus genome is the E1 region, isthat the expression of E1 protein (from the complementing cell) mayincrease expression of the protein of interest (from the adenovirustransgene), especially when the protein of interest is under control ofcertain promoters that can be transactivated by E1A, e.g., the CMVpromoter. In certain embodiments, the second type of complementing cellsas used in step c) of the invention are HEK293 cells. In a particularlypreferred embodiment, the complementing cells in step c) are PER.C6cells. PER.C6 cells for the purpose of the invention means cells from anupstream or downstream passage or a descendent of an upstream ordownstream passage of cells as deposited under ECACC no. 96022940, i.e.,having the characteristics of those cells. PER.C6 cells have severaladvantages over other E1-complementing cells, for instance with regardto scalability, safety, ease of handling, and the wide expertise thathas been built up with this cell line over the course of many programsfor production of pharmaceutical materials using these cells by variouscompanies. PER.C6 cells have for instance been cultured at 20,000 literscale, demonstrating its applicability for large scale production.Extensive characterization of the cell line, and administration tonumerous subjects of several different pharmaceutical products producedin this cell line without any cell line related adverse event, hasdemonstrated its safety for pharmaceutical purposes. We furtherdemonstrate herein that the yields of recombinant protein produced usingthe method of the invention are surprisingly high.

The second region of the adenovirus genome may be chosen from any regionthat is required for adenovirus genome replication or adenovirusparticle formation, and can be E1 (only in embodiments where the firstregion is not the E1 region), but more typically is chosen from otherparts of the early region of the adenovirus genome, e.g., E2A, E2B (AdDNA polymerase) or E4. These regions are required for adenovirus genomereplication. Recombinant adenovirus vectors with deletions in E1 andE2A, E2B or E4, as well as cells that complement such vectors have beendescribed before, and are thus available (see, e.g., U.S. Pat. Nos.6,063,622, 6,482,616 and 6,133,028, incorporated by reference).

In further embodiments, the second region of the adenovirus genome is aregion that is not required for replication, but is required forparticle formation, e.g., a late gene such as 100K and/or 33K (bothencoded by the L4 region, required for correct splicing of the majorlate transcription unit (MLTU) and therefore for late gene expression;Farley et al, 2004). One potential advantage of such embodiments is thatthe adenovirus particles used for infection in step c) can replicateinside the complementing cell, so that the amount of template DNA fromwhich protein is expressed is amplified and thus expression of theprotein of interest may be further increased, while at the same time nonew viral particles can be formed. Adenovirus vectors with deletions ofE1 and the 100K gene and complementation cells for such vectors havebeen described and are thus available (Hodges et al, 2001; U.S. Pat. No.6,328,958, incorporated by reference). The same is true for pTP and IVa2proteins (U.S. Pat. No. 6,328,958), and these can thus also be used asthe second region of the adenovirus genome in further embodiments of theinvention. Further possibilities for the second region of the adenovirusgenome according to the invention include hexon, 51K/55K, andcombinations of any of the above. The DNA sequence of the Ad5 genome andthe encoded proteins therein are completely known (Genbank M73260), andthe skilled person can thus generate the desired adenovirus vectors, andthe desired complementing cell lines, using routine molecular biologymethods.

In certain embodiments, the second region of the adenovirus genome isthe E2A region. Adenovirus with deletions in both E1 and E2A has beendescribed, and cell lines expressing E2A have also been describedpreviously, e.g., in U.S. Pat. No. 6,395,519, incorporated in itsentirety herein by reference. Such cell lines may be based on PER.C6cells (that express the E1 region), wherein the E2A region has beenintroduced and integrated into the genome. Such cell lines may also bebased on other E1-complementing cell lines such as HEK293, wherein theE2A region has been introduced. In certain embodiments, a temperaturesensitive E2A protein is expressed in the first type of complementingcells used in step b) of the invention, for instance the ts125 E2Amutant (see U.S. Pat. No. 6,395,519). In other embodiments, wild typeE2A is expressed and regulated by an inducible promoter (see, e.g., U.S.Pat. No. 6,482,616, incorporated by reference herein). Thus, the firsttype of complementing cell lines that can be used in step b) of theinvention have also been described and are available to the skilledperson. In certain preferred embodiments, the first type ofcomplementing cells used in step b) of the invention are PER.C6 cellsthat express the ts125 E2A protein, which cells are referred to hereinas PER.E2A or PER.C6.E2A (see U.S. Pat. No. 6,395,519 for generation ofthis cell line). E1-deleted adenovirus can be propagated in these cells,and also E1/E2A-deleted adenovirus (adenovirus with deletions in both E1and E2A) can be propagated in these cells, while PER.C6 cells (withoutE2A) can only be used to propagate E1-deleted adenovirus. E1/E2A-deletedadenovirus is most efficiently propagated in PER.E2A cells at 34° C.,although temperatures of 32° C.-37° C. can be used. In step b) of theinvention, recombinant adenovirus particles are formed upon replicationand particle formation in the first type of complementing cell. Theseparticles are subsequently used for infection of the second type ofcomplementing cell, which will not complement all deficiencies in theadenoviral genome, but will allow for expression of the recombinantprotein of interest that is encoded by the transgene in the adenovirusgenome.

The recombinant adenovirus may in certain embodiments further have adeletion in E3, but since this region is not required for adenovirusgenome replication or adenovirus particle formation, it needs not becomplemented. Deletion of the E3 region or part thereof can beadvantageous if the transgene is too large to be accommodated by theadenovirus having only deletions in the first and second regions of theadenovirus genome, as is known to the skilled person. Deletion of the E3region or part thereof from a recombinant adenovirus genome can be doneby routine molecular biology methods known to the person skilled in theart. For the methods of the invention, adenovirus containing the E3region, as well as adenovirus wherein E3 has been deleted, have beenused and gave similar results.

The invention in one preferred embodiment thus provides a method forproducing a protein of interest, the method comprising: a) providing arecombinant adenoviral vector comprising nucleic acid encoding theprotein of interest under control of a promoter, wherein the adenoviralvector has deletions in the E1 and the E2A regions of the adenovirusgenome, b) propagating the adenoviral vector in complementing cells thatexpress adenoviral E1 protein and adenoviral E2A protein, to obtainrecombinant adenovirus particles, c) infecting a culture ofcomplementing cells that express adenoviral E1 protein but notadenoviral E2A protein with the recombinant adenovirus particles, toproduce the protein of interest, and d) harvesting the protein ofinterest.

The serotype of the adenovirus for the invention is not material, andthe invention can thus be practiced with adenovirus of any serotype,including adenovirus type 2, 5, 7, 11, 26, 35, 49, 50, 51, or a chimericadenovirus with a genome comprising parts derived from differentserotypes, and the like. It can further be an adenovirus from differentspecies, e.g., bovine, ovine, canine, etc, but in preferred embodimentsis a human type adenovirus. Complementing cells for several adenovirusserotypes and adenovirus species have been previously described and arethus available for use according to the invention. In certainembodiments, the adenovirus is a human adenovirus type 5 (Ad5).

Recombinant adenovirus genome can be routinely prepared using standardtechnology, known to the skilled person (e.g., Hosfield and Eldridge,Strategies 13: 100-102, Stratagene). One exemplary method uses twoplasmids that are introduced into the first type of complementing cellto form the adenoviral genome via homologous recombination (see e.g.,U.S. Pat. No. 6,878,549, incorporated by reference herein). Anothermethod uses two plasmids that are first formed into an adenoviral genomein bacteria or yeast, from which the adenovirus genome is isolated andsubsequently used for introduction into the first type of packaging cell(see e.g., He et al, 1998, U.S. Pat. No. 5,922,576, incorporated byreference herein). Such a system is commercially available (described byHosfield and Eldridge in Strategies 13: 100-102, Stratagene). A firstplasmid in these methods suitably contains the left end of theadenovirus genome, wherein E1 has been deleted and is or has beenreplaced by a transgene that encodes the protein of interest (e.g., inthe form of an expression cassette: a promoter operably linked tonucleic acid encoding the protein of interest, and usually atranscription terminator/polyadenylation signal). The second plasmid isusually much larger and suitably is a cosmid (see e.g., U.S. Pat. No.6,878,549). It contains the remainder of the adenovirus genome and hasthe desired deletions, e.g., an E2A deletion, and optionally an E3deletion. Upon recombination these plasmids form the recombinantadenoviral vector comprising the nucleic acid encoding the protein ofinterest under control of a promoter, of step a) of the invention.Introduction of this vector in the first complementing cells leads topropagation and adenovirus particle generation (step b) of theinvention). Cloning of nucleic acid encoding the protein of interestinto the adenovirus genome can be done by routine methods, for instancein plasmids containing the left end of the adenovirus genome with adeletion in E1, often referred to as adapter plasmids (e.g., U.S. Pat.No. 6,878,549).

In steps b) and c) of the invention, the complementing cells arecultured in suitable culture medium, using routine methods known to theskilled person. The protein of interest as produced in step c) ispreferably secreted into the culture medium. Step d) comprises theharvesting of the protein of interest, and can be performed according tomethods known to the skilled person. Harvesting comprises separating thecomplementing cells from the culture medium, e.g., by centrifugation,filtration, or other methods. The culture medium (supernatant) containsthe protein of interest, and can be used directly for certainapplications (e.g., testing production levels, product qualityanalysis), but more typically the protein of interest is furtherpurified from the cell culture supernatant. This can be done usingmethods known to the skilled person, and dependent from the protein ofinterest. Methods to purify proteins of interest from a biologicalmatrix such as a cell culture supernatant are available, and usuallycomprise one or more of centrifugation, filtration, chromatography, andthe like. Various useful chromatographic methods are available to purifyproteins, including but not limited to anion exchange chromatography,cation exchange chromatography, hydrophobic interaction chromatography,affinity chromatography (e.g., antibody purification on protA columns),size exclusion chromatography, hydroxyapatite chromatography, etc. Theskilled person will use methods that are most appropriate forpurification of the protein of interest to the desired purity. One ormore known virus inactivation steps may be included in the purificationprocedure if the protein is to be administered to humans.

The protein of interest can be any protein. In certain embodiments, theprotein of interest is selected from the group consisting of anantibody, erythropoietin, a blood clotting factor such as Factor VIII,Factor IX, Factor X, Factor XIII, Factor V, Factor VII, or variantsthereof, a hormone, a viral protein other than an adenovirus protein,such as an influenza protein, such as hemaglutinin (HA) or neuraminidase(NA), etc. In certain embodiments, the protein of interest is a humanprotein. In certain embodiments, the protein is a secreted protein.Non-limiting examples of a protein of interest according to theinvention are enzymes, hormones, immunoglobulin chains, therapeuticproteins like anti-cancer proteins, blood coagulation proteins,multi-functional proteins, diagnostic proteins, or proteins or fragmentsthereof useful for vaccination purposes, all known to the person skilledin the art. In certain embodiments, the protein of interest comprisespost-translational modifications such as glycosylation, i.e., it is aglycoprotein. In certain embodiments, it comprises at least a variablepart of an immunoglobulin heavy or light chain. In certain embodiments,the protein of interest is an antibody. In case where the protein ofinterest comprises more than one subunit, nucleic acid encoding eachsubunit can be suitably cloned in an adenovirus vector in the form of amulticistronic transcription unit, e.g., by using an internal ribosomeentry site (IRES), according to methods and using means well known tothe person skilled in the art. As used herein, an “internal ribosomeentry site” or “IRES” refers to an element that promotes direct internalribosome entry to the initiation codon of a cistron (a protein encodingregion), thereby leading to the cap-independent translation of the gene.IRES sequences and use thereof for expression are well known to theperson skilled in the art. See e.g., Jackson R J, Howell M T, Kaminski A(1990) Trends Biochem Sci 15 (12): 477-83), Jackson R J and Kaminski, A.(1995) RNA 1 (10): 985-1000, Martinez-Salas, 1999, and Rees et al, 1996.In other embodiments, instead of using an IRES, two or more proteins orsubunits are expressed from separate promoters, and both expressionunits (promoter+sequence encoding protein/subunit of interest) isincorporated into a single adenoviral vector. In such embodiments, itmay be beneficial to use different promoters for each of theproteins/subunits, in order to increase the stability of the resultingadenovirus. In certain embodiments, one promoter is a CMV promoter.Alternatively, to express more than one protein or more than one subunitof a multimeric protein such as an antibody, cells in step c) may beco-infected with two or more recombinant adenovirus vectors, each codingfor a different protein or subunit. Experiments with heavy and lightchain of an antibody each encoded on a different adenovirus vector andco-infection of these vectors into the second type of complementingcells according to step c) of the invention showed that this alsoresulted in expression of antibodies, and thus this method can be usedas an alternative to expressing both subunits from a single adenovirusvector using an IRES between heavy and light chain.

Nucleic acid encoding a protein of interest is typically available, forinstance in the form of cDNA, genomic DNA or synthetic DNA, and can becloned in the recombinant adenoviral vector. The nucleic acid encodingthe protein of interest preferably is cloned in operable associationwith a promoter that can drive transcription of the nucleic acid, allaccording to standard and routine methods known to the skilled person(see, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual, 2nd edition, 1989).

Co-expression of an enzyme involved in glycosylation, e.g., asialyltransferase, e.g., an alfa-2,6-sialyltransferase or analfa-2,3-sialyltransferase, with the protein of interest can be achievedusing methods of the invention. Methods and means for over-expression ofenzymes involved in post-translational modification, in particularalfa-2,6-sialyltransferase or alfa-2,3-sialyltransferase, in PER.C6cells have been previously described, and were shown to increasesialylation of recombinant proteins produced in PER.C6 cells. See, e.g.,US 2005/0164386 and US 2005/0181359, both incorporated herein byreference. A suitable α-2,6-sialyltransferase is a humanα-2,6-sialyltransferase, the sequence of which was described byGrundmann et al, 1990. In certain embodiments, the second type ofcomplementing cells express recombinant nucleic acid encoding analfa-2,3-sialyltransferase, for instance under control of a heterologouspromoter. The alfa-2,3-sialyltransferase may for instance be a humanα-2,3-sialyltransferase, known as SIAT4C or STZ (Genbank accessionnumber L23767, see also U.S. Pat. No. 5,494,790). In certainembodiments, sialyltransferase may be over-expressed by integration ofthe DNA encoding the sialyltransferase into the genome of the secondtype of complementing cells. In other and preferred embodiments,sialyltransferase is co-expressed with the protein of interest byincluding the nucleic acid encoding the sialyltransferase in theadenovirus vector, e.g., in operable linkage with an IRES, behind thenucleic acid encoding the protein of interest, or in operable linkagewith a separate promoter. Infection of the second type of complementingcells in step c) of the invention with such an adenovirus will ensureexpression of both the protein of interest and of the sialyltransferase.Alternatively, the sialyltransferase may be co-expressed from adifferent adenoviral vector that is co-infected into the second type ofcomplementing cell in step c) of the invention, as described supra.

In general, the production of a recombinant protein in a host cellcomprises the introduction of nucleic acid in expressible format intothe host cell, culturing the cells under conditions conducive toexpression of the nucleic acid and allowing expression of the nucleicacid in the cells. For the purpose of this application “express,”“expressing” or “expression” refers to the transcription and translationof a gene encoding a protein.

Nucleic acid encoding a protein in expressible format may be in the formof an expression cassette, and usually requires sequences capable ofbringing about expression of the nucleic acid, such as enhancer(s),promoter, polyadenylation signal, and the like. Several promoters can beused for expression of recombinant nucleic acid, and these may compriseviral, mammalian, synthetic promoters, and the like. In certainembodiments, a promoter driving the expression of the nucleic acid ofinterest is the cytomegalovirus (CMV) immediate early promoter, forinstance comprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter, as this promoter has been shown to give highexpression levels in cells expressing E1A of an adenovirus such as thecells of the invention (see, e.g., WO 03/051927). The nucleic acidencoding the protein of interest may be a genomic DNA, a cDNA, syntheticDNA, a combination of these, etc. In certain embodiments, the nucleicacid encoding the protein of interest is a cDNA. If desired, one or moreartificial or natural introns may be re-inserted into the cDNA (see,e.g., EP 1283263). It is also possible to remove cryptic splice sites toenhance the expression (see, e.g., U.S. Pat. No. 6,642,028). Codons maybe optimized if desired to improve expression (see, e.g., U.S. Pat. No.6,114,148).

Some well-known and much used promoters for expression in eukaryoticcells comprise promoters derived from viruses, such as adenovirus, e.g.,the E1A promoter, promoters derived from cytomegalovirus (CMV), such asthe CMV immediate early (IE) promoter (referred to herein as the CMVpromoter) (obtainable, for instance, from pcDNA, Invitrogen), promotersderived from Simian Virus 40 (SV40) (Das et al., 1985), and the like.Suitable promoters can also be derived from eukaryotic cells, such asmethallothionein (MT) promoters, elongation factor 1α (EF-1α) promoter(Gill et al., 2001), ubiquitin C or UB6 promoter (Gill et al., 2001;Schorpp et al., 1996), actin promoter, an immunoglobulin promoter, heatshock promoters, and the like. Some preferred promoters for obtainingexpression in eukaryotic cells, which are suitable promoters in theinvention, are the CMV-promoter, a mammalian EF1-alpha promoter, amammalian ubiquitin promoter such as a ubiquitin C promoter, or a SV40promoter (e.g., obtainable from pIRES, cat. no. 631605, BD Sciences).Testing for promoter function and strength of a promoter is a matter ofroutine for a person skilled in the art, and in general may for instanceencompass cloning a test gene such as lacZ, luciferase, GFP, etc.,behind the promoter sequence, and test for expression of the test gene.Of course, promoters may be altered by deletion, addition, mutation ofsequences therein, and tested for functionality, to find new,attenuated, or improved promoter sequences. Strong promoters that givehigh transcription levels in the second complementing cells of theinvention are preferred.

Introduction of the nucleic acid that is to be expressed in a cell, canbe done by one of several methods, which as such are known to the personskilled in the art, also dependent on the format of the nucleic acid tobe introduced. The methods include but are not limited to transfection,infection, injection, transformation, and the like. For the invention, arecombinant adenovirus is used to infect the second type ofcomplementing cells as host cells for subsequent protein production.Infection takes place by mixing the recombinant adenovirus with thecells, e.g., in a cell culture medium wherein the cells are cultured.

The terms “cell culture medium” and “culture medium” refer to a nutrientsolution used for growing mammalian cells that typically provides atleast one component from one or more of the following categories: 1) anenergy source, usually in the form of a carbohydrate such as glucose; 2)all essential amino acids, and usually the basic set of twenty aminoacids plus cysteine; 3) vitamins and/or other organic compounds requiredat low concentrations; 4) free fatty acids; and 5) trace elements, wheretrace elements are defined as inorganic compounds or naturally occurringelements that are typically required at very low concentrations, usuallyin the micromolar range. The nutrient solution may optionally besupplemented with one or more components from any of the followingcategories: 1) hormones and other growth factors as, for example,insulin, transferrin, and epidermal growth factor; 2) salts and buffersas, for example, calcium, magnesium, and phosphate; 3) nucleosides andbases such as, for example, adenosine, thymidine, and hypoxanthine; and4) protein and tissue hydrolysates. Cell culture media are availablefrom various vendors, and serum-free culture media are nowadays oftenused for cell culture, because they are more defined than mediacontaining serum. The cells of the invention grow well inserum-containing media as well as in serum-free media. Usually some timeis required to adapt the cells from a serum containing medium, such asDMEM+FBS, to a serum-free medium. Examples of serum-free culture mediumthat are suitable for use in the invention are EX-CELL™ VPRO medium (JRHBiosciences) and AEM medium (Invitrogen). The cells of the invention ingeneral grow adherently in serum-containing media, but are veryproficient in growing in suspension to high cell densities (10×10⁶cells/ml and higher) in serum-free culture media, which means that theydo not need a surface to adhere to, but remain relatively free from eachother and from the walls of the culture vessel during most of the time.Processes for culturing the cells of the invention to high densitiesand/or for obtaining very high product yields from these cells have beendescribed (WO 2004/099396), incorporated herein by reference. Culturinga cell is done to enable it to metabolize, and/or grow and/or divideand/or produce recombinant proteins of interest. This can beaccomplished by methods well known to persons skilled in the art, andincludes but is not limited to providing nutrients for the cell. Themethods comprise growth adhering to surfaces, growth in suspension, orcombinations thereof. Culturing can be done for instance in dishes,roller bottles or in bioreactors, using batch, fed-batch, continuoussystems such as perfusion systems, and the like. In order to achievelarge scale (continuous) production of recombinant proteins through cellculture it is preferred in the art to have cells capable of growing insuspension, and it is preferred to have cells capable of being culturedin the absence of animal- or human-derived serum or animal- orhuman-derived serum components. The conditions for growing ormultiplying cells (see, e.g., Tissue Culture, Academic Press, Kruse andPaterson, editors (1973)) and the conditions for expression of therecombinant product are known to the person skilled in the art. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of mammalian cell cultures can be found in MammalianCell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press,1991).

For small scale experiments, adherent cultures can be used. In preferredembodiments however, the second type of complementing cells in step c)of the invention are in suspension. Suspension cultures are scalable,which is an advantage of such embodiments. PER.C6 cells are verysuitable for suspension culture, and can be cultured in suspension tovery high cell densities. HEK293 cells can also be cultured insuspension. Thus, in preferred embodiments, the culture of complementingcells in step c) of the invention is a suspension culture.

The protein of interest may be produced by growing the cells of theinvention that express the desired protein under a variety of cellculture conditions. For instance, cell culture procedures for the largeor small-scale production of proteins are potentially useful within thecontext of the invention. Procedures including, but not limited to, afluidized bed bioreactor, hollow fiber bioreactor, roller bottleculture, or stirred tank bioreactor system may be used, in the later twosystems, with or without microcarriers, and operated alternatively in abatch, fed-batch, or continuous mode. In preferred embodiments, thecells are in suspension. For simplicity and economic reasons, it ispreferred to use a batch process for culturing the cells for theinvention. However, if very high cell densities are desired prior toinfection, it can be useful to feed culture medium or certain nutrientsto the cells in certain embodiments, using methods that have beendescribed previously (fed-batch and perfusion cultures for PER.C6 cells;e.g., WO 2004/099396 and WO 2005/095578, both incorporated by referenceherein).

The protein of interest preferably is secreted into the culture medium.Naturally secreted proteins contain secretion signals that bring aboutsecretion of the produced proteins. The sequences of secretion signalsare known, and can be engineered into non-naturally secreted proteins ifdesired. Further, sequences that prevent secretion, e.g., transmembranedomains of receptors, can often be removed so that soluble proteins areobtained. In a preferred embodiment, the expressed protein is collected(isolated), preferably from the culture medium. The protein of interestthus preferably is recovered from the culture medium as a secretedpolypeptide. For example, as a first step, the culture medium iscentrifuged to remove particulate cell debris. The polypeptidethereafter is preferably purified from contaminant soluble proteins andpolypeptides, e.g., by filtration, column chromatography, etc, bymethods generally known to the person skilled in the art. Suitablepurification steps include methods which were known in the art can beused to maximize the yield of a pure, stable and highly active productand are selected from immunoaffinity chromatography, anion exchangechromatography, size exclusion chromatography, etc., and combinationsthereof. In certain embodiments, the harvesting of the protein ofinterest includes at least one chromatography step. The chromatographycan be performed in batch, columns, or in other formats, e.g., usingmembranes that include functional groups. In certain embodiments, theprotein of interest is purified to at least 90%, preferably at least95%, still more preferably at least 99% purity. In particularlypreferred embodiments, the protein of interest is purified andformulated into a pharmaceutical composition that is suitable foradministration to human subjects, according to methods known to theskilled person.

Since adenovirus particles are much larger than proteins, asize-exclusion step during purification of the protein will alreadyensure removal of any inadvertently present adenovirus particles.However, to be on the safe side and overcome the potential problems ofpossible infectious contaminations in the purified protein samples or inthe product directly obtained from the cell culture supernatantcontaining the secreted recombinant protein of choice, the samplesand/or the culture supernatant might be treated with procedures forvirus inactivation including heat treatment (dry or in liquid state,with or without the addition of chemical substances including proteaseinhibitors), which procedures are known to the skilled person. Aftervirus inactivation a further purifying step for removing the chemicalsubstances may be necessary, as known to the skilled person. The PER.C6cells of the invention are available free of adventitious virus and TSEand a well-documented history is available for these cells, so that theycan be used to produce proteins in a safe manner.

In step c) of the method of the invention, the second type ofcomplementing cells are infected with recombinant adenovirus particles.This can suitably be performed along a very wide range of multiplicityof infections (moi), generally the moi is between 1 and 10000 virusparticles (vp)/cell. It has been surprisingly found herein that lowermoi's work as good or better than higher moi's, so that in preferredembodiments the moi is between 1 and 2000 vp/cell, more preferablybetween 1 and 1000 vp/cell, more preferably between 1 and 200 vp/cell,still more preferably between 1 and 100 vp/cell, typically between 10and 100 vp/cell, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, or 90vp/cell. The advantage of the use of lower moi's (100 vp/cell or lower)is that less virus is required, so that step b) of the invention becomesless burdensome: more cells in step c) can be infected with materialobtained in step b). This makes the system economically more attractivesince less culture media, less time, and smaller reaction vessels arerequired for step b) then when high moi's would be required for step c).One infectious unit (IU) generally equals about 1-100 vp (depending onvariations in batch quality), more typically equals between 5-20 vp. Incertain embodiments of the invention, the second type of complementingcells are infected with about between 1-10 IU of the recombinantadenovirus particles obtained in step b) of the invention.

For the methods of the invention, the moi does not necessarily have tobe determined prior to infection of step c). It is for instance possibleto establish an estimation of the moi for certain conditions with aspecific vector, and infect the culture of the second type ofcomplementing cells with an amount of virus based on such estimation. Itis also possible to infect several cultures of the second type ofcomplementing cells each with a different amount of virus particles, andempirically find out which worked best when the protein is harvested. Ifdesired, the experiment may be scaled up using the findings of thisempirical determination.

In certain embodiments, the recombinant adenovirus particles of step b)of the invention are purified first, and subsequently the purifiedrecombinant adenovirus particles are used to infect the second type ofcomplementing cells in step c). Purification of recombinant adenovirusparticles can be done by a variety of previously described methods,including CsCl centrifugation, filtration and chromatography, and thelike (see, e.g., WO 2005/080556 and references therein, and WO2006/108707 and references therein, for a variety of useful methods forpurification of recombinant adenovirus, all incorporated by referenceherein). One advantage of such a method is the high degree of controlover the virus quality and quantity that is achieved upon purification,before the virus is used in step c) of the invention.

However, purification of the recombinant adenovirus particles is alengthy process typically taking several weeks to months. Therefore itis highly preferable to use the recombinant virus particles of step b)immediately, i.e., without further purification steps, for the infectionin step c) of the invention. It is shown herein that this is possible,and surprisingly gives very good results. The results with the so-called‘crude’ virus, i.e., culture medium of step b) from which the first typeof complementing cells have been removed, e.g., by centrifugation, areat least as good as with purified virus, regarding quantity and qualityof the protein of interest obtained, while the methods wherein the crudevirus is used are significantly shorter. The advantage is thus that themethod of the invention becomes very fast if no virus purification stepsare performed. The present methods allow use of such fast methods, andno virus purification, nor exact determination of the moi, is requiredto obtain high yields of intact protein of interest in a very fastprocess. Therefore, in certain embodiments, the recombinant adenovirusparticles in step b) are obtained in a culture medium, and the culturemedium containing the recombinant adenovirus particles is separated fromthe first type of complementing cells to obtain a crude adenovirusparticle composition, and the crude adenovirus particle composition isused for the infection of the second type of complementing cells in stepc). In advantageous embodiments, a freeze-thaw cycle is performed withthe culture of step b) to release more of the recombinant adenovirusparticles. This increases the amount of particles that can be used forstep c), and has the additional advantage to kill the first type ofcomplementing cells, and thus further decreases the chance that suchcells inadvertently end up in the culture of step c). The separation ofthe medium and the virus particles from the cells can be done usingsimple methods such as centrifugation or filtration, and the like,making use of the large size and weight differences between cells andvirus particles, in a manner known to the skilled person.

It will be clear that step b) can be performed first and independentlyfrom step c), to obtain the recombinant adenovirus particle composition.The recombinant adenovirus can then for instance optionally be storedand used for step c) at a later time if desired. Therefore, step c) is apossible embodiment of the invention as well. The invention thereforefurther provides a method for producing a protein of interest, themethod comprising: infecting a culture of complementing cells with arecombinant adenoviral particle that has a genome comprising nucleicacid encoding the protein of interest under control of a promoter,wherein the genome has deletions in a first region and in a secondregion of the adenovirus genome, wherein each of the first region andthe second region is required for adenoviral genome replication and/oradenovirus particle formation, wherein the complementing cells expressprotein from the first region of the adenovirus genome but not proteinfrom the second region of the adenovirus genome, to produce the proteinof interest, and harvesting the protein of interest. In preferredembodiments hereof, the first region of the adenovirus is E1. In certainembodiments, the second region of the adenovirus is E2A. Preferredcomplementing cells of this aspect express adenovirus E1 protein, suchas HEK293 cells, 911 cells, PER.C6 cells, and the like. The mostpreferred complementing cells for this aspect are PER.C6 cells.

To illustrate the invention, the following examples are provided, notintended to limit the scope of the invention. The human erythropoietin(EPO) molecule contains four carbohydrate chains. Three containN-linkages to asparagines, and one contains an O-linkage to a serineresidue. The importance of glycosylation in the biological activity ofEPO has been well documented (Delorme et al., 1992; Yamaguchi et al.,1991). The cDNA encoding human EPO was cloned and expressed in PER.C6cells and PER.C6/E2A cells, expression was shown, and the glycosylationpattern was analyzed.

The practice of this invention will employ, unless otherwise indicated,conventional techniques of immunology, molecular biology, microbiology,cell biology, and recombinant DNA, which are within the skill of theart. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual, 2nd edition, 1989; Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds, 1987; the series Methods inEnzymology (Academic Press, Inc.); PCR2: A Practical Approach, M. J.MacPherson, B. D. Hams, G. R. Taylor, eds, 1995; Antibodies: ALaboratory Manual, Harlow and Lane, eds, 1988.

EXAMPLES Example 1 Construction of Basic Expression Vectors

Plasmid pcDNA3.1/Hygro(−) (Invitrogen) was digested with NruI and EcoRV,dephosphorylated at the 5′ termini by Shrimp Alkaline Phosphatase (SAP,GIBCO Life Tech.) and the plasmid fragment lacking the immediate earlyenhancer and promoter from CMV was purified from gel. Plasmid pAdApt(Crucell N V of Leiden, N L), containing the full length CMVenhancer/promoter (−735 to +95) next to overlapping Adeno-derivedsequences to produce recombinant adenovirus, was digested with AvrII,filled in with Klenow polymerase and digested with HpaI; the fragmentcontaining the CMV enhancer and promoter was purified over agarose gel.This CMV enhancer and promoter fragment was ligated bluntiblunt to theNruI/EcoRV fragment from pcDNA3.1/Hygro(−). The resulting plasmid wasdesignated pcDNA2000/Hyg(−).

Plasmid pcDNA2000/Hyg(−) was digested with PmlI, and the linearizedplasmid lacking the Hygromycin resistance marker gene was purified fromgel and religated. The resulting plasmid was designated pcDNA2000.Plasmid pcDNA2000 was digested with PmlI and dephosphorylated by SAP atboth termini. Plasmid pIG-GC9 containing the wild-type human DHFR cDNA(Havenga et al., 1998) was used to obtain the wild-type DHFR-gene bypolymerase chain reaction (PCR) with introduced, noncoding PmlI sitesupstream and down stream of the cDNA. PCR primers that were used wereDHFR up: 5′-GAT CCA CGT GAG ATC TCC ACC ATG GTT GGT TCG CTA AAC TG-3′(SEQ ID NO:1), corresponding to the SEQUENCE LISTING of U.S. patentapplication Ser. No. 10/234,007, now U.S. Pat. No. 7,132,280 (hereafterreferred to as ‘the '007 application’) of Bout et al., the contents ofthe entirety of which are incorporated by this reference) and DHFR down:5′-GAT CCA CGT GAG ATC TTT AAT CAT TCT TCT CAT ATAC-3′ (SEQ ID NO:2)corresponding to the incorporated '007 application. The PCR-product wasdigested with PmlI and used for ligation into pcDNA2000 (digested withPmlI, and dephosphorylated by SAP) to obtain pcDNA2000/DHFRwt (FIG. 1 ofthe incorporated '007 application). Wild-type sequences and correctlyused cloning sites were confirmed by double stranded sequencing.Moreover, a mutant version of the human DHFR gene (DHFRm) was used toreach a 10,000-fold higher resistance to methotrexate in PER.C6 andPER.C6/E2A by selection of a possible integration of the transgene in agenomic region with high transcriptional activity. This mutant carriesan amino acid substitution in position 32 (phenylalanine to serine) andposition 159 (leucine to proline) introduced by the PCR procedure. PCRon plasmid pIG-GC12 (Havenga et al., 1998) was used to obtain the mutantversion of human DHFR. Cloning of this mutant is comparable to wild-typeDHFR. The plasmid obtained with mutant DHFR was designatedpcDNA2000/DHFRm.

pIPspAdapt 6 (Galapagos Genomics of Belgium) was digested with AgeI andBamHI restriction enzymes. The resulting polylinker fragment has thefollowing sequence: 5′-ACC GGT GAA TTC GGC GCG CCG TCG ACG ATA TCG ATCGGA CCG ACG CGT TCG CGA GCG GCC GCA ATT CGC TAG CGT TAA CGG ATC C -3′(SEQ ID NO:3) corresponding to the incorporated '007 application. Theused AgeI and BamHI recognition sites are underlined. This fragmentcontains several unique restriction enzyme recognition sites and waspurified over agarose gel and ligated to an AgeI/BamHI-digested andagarose gel-purified pcDNA2000/DHFRwt plasmid. The resulting vector wasnamed pcDNA2001/DHFRwt (FIG. 2 of the incorporated '007 application).

pIPspAdapt7 (Galapagos of Belgium) is digested with AgeI and BamHIrestriction enzymes and has the following sequence: 5′-ACC GGT GAA TTGCGG CCG CTC GCG AAC GCG TCG GTC CGT ATC GAT ATC GTC GAC GGC GCG CCG AATTCG CTA GCG TTA ACG GAT CC-3′ (SEQ ID NO:4) corresponding to theincorporated '007 application. The used AgeI and BamHI recognition sitesare underlined in the incorporated '007 application. The polylinkerfragment contains several unique restriction enzyme recognition sites(different from pIPspAdapt6), which are purified over agarose gel andligated to an AgeI/BamHI-digested and agarose gel-purifiedpcDNA2000/DHFRwt. This results in pcDNA2002/DHFRwt (FIG. 3 of theincorporated '007 application).

pcDNA2000/DHFRwt was partially digested with restriction enzyme PvuII.There are two PvuII sites present in this plasmid and cloning wasperformed into the site between the SV40 poly(A) and ColE1, not thePvuII site down stream of the BGH poly(A). A single site-digestedmixture of plasmid was dephosphorylated with SAP and blunted with Klenowenzyme and purified over agarose gel. pcDNA2000/DHFRwt was digested withMunI and PvuII restriction enzymes and filled in with Klenow and freenucleotides to have both ends blunted. The resulting CMVpromoter-linker-BGH poly(A)-containing fragment was isolated over geland separated from the vector. This fragment was ligated into thepartially digested and dephosphorylated vector and checked fororientation and insertion site. The resulting plasmid was namedpcDNAs3000/DHFRwt (FIG. 4 of the incorporated '007 application).

Example 2 Construction of EPO Expression Vectors

The full length human EPO cDNA was cloned, employing oligonucleotideprimers EPO-START: 5′ AAA AAG GAT CCG CCA CCA TGG GGG TGC ACG AAT GTCCTG CCT G-3′ (SEQ ID NO:5) corresponding to the incorporated '007application and EPO-STOP: 5′-AAA AAG GAT CCT CAT CTG TCC CCT GTC CTG CAGGCC TC-3′ (SEQ ID NO:6) corresponding to the incorporated '007application (Cambridge Bioscience Ltd.) in a PCR on a human adult livercDNA library. The amplified fragment was cloned into pUC18 linearizedwith BamHI. Sequence was checked by double stranded sequencing. Thisplasmid containing the EPO cDNA in pUC18 was digested with BamHI and theEPO insert was purified from agarose gel. Plasmids pcDNA2000/DHFRwt andpcDNA2000/DHFRm were linearized with BamHI and dephosphorylated at the5′ overhang by SAP, and the plasmids were purified from agarose gel. TheEPO cDNA fragment was ligated into the BamHI sites of pcDNA2000/DHFRwtand pcDNA2000/DHFRm; the resulting plasmids were designatedpEPO2000/DHFRwt (FIG. 5 of the incorporated '007 application) andpEPO2000/DHFRm.

The plasmid pMLPI.TK (described in PCT International Patent PublicationNo. WO 97/00326) is an example of an adapter plasmid designed for use incombination with improved packaging cell lines like PER.C6 (described inPCT International Patent Publication No. WO 97/00326 and U.S. Pat. No.6,033,908 to Bout et al. (Mar. 7, 2000), the contents of both of whichare incorporated by this reference). First, a PCR fragment was generatedfrom pZipDMo+PyF101(N—) template DNA (described in International PatentApplication No. PCT/NL96/00195) with the following primers: LTR-1(5′-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3′(SEQ ID NO:7) corresponding to the incorporated '007 application andLTR-2 (5′-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AACCGG GCG ACT CAG TCA ATC G-3′ (SEQ ID NO:8) corresponding to theincorporated '007 application). The PCR product was then digested withBamHI and ligated into pMLP10 (Levrero et al., 1991), that was digestedwith PvuII and BamHI, thereby generating vector pLTR10. This vectorcontains adenoviral sequences from bp 1 up to bp 454 followed by apromoter consisting of a part of the Mo-MuLV LTR having its wild-typeenhancer sequences replaced by the enhancer from a mutant polyoma virus(PyF101). The promoter fragment was designated L420. Next, the codingregion of the murine HSA gene was inserted. pLTR10 was digested withBstBI followed by Klenow treatment and digestion with NcoI. The HSA genewas obtained by PCR amplification on pUC18-HSA (Kay et al., 1990, usingthe following primers: HSAI (5′-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3′(SEQ ID NO:9) corresponding to the incorporated '007 application) andHSA2 (5′-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAGAA-3′ (SEQ ID NO:10) corresponding to the incorporated '007application). The 269 bp PCR fragment was subcloned in a shuttle vectorusing NcoI and BglII sites. Sequencing confirmed incorporation of thecorrect coding sequence of the HSA gene, but with an extra TAG insertiondirectly following the TAG stop codon. The coding region of the HSAgene, including the TAG duplication, was then excised as a NcoI/SalIfragment and cloned into a 3.5 kb NcoI/BstBI cut pLTR10, resulting inpLTR-HSA10. This plasmid was digested with EcoRI and BamHI, after whichthe fragment, containing the left ITR, the packaging signal, the L420promoter and the HSA gene, was inserted into vector pMLPI.TK digestedwith the same enzymes and thereby replacing the promoter and genesequences, resulting in the new adapter plasmid pAd5/L420-HSA.

The pAd5/L420-HSA plasmid was digested with AvrII and BglII followed bytreatment with Klenow and ligated to a blunt 1570 bp fragment frompcDNA1/amp (Invitrogen) obtained by digestion with HhaI and AvrIIfollowed by treatment with T4 DNA polymerase. This adapter plasmid wasnamed pAd5/CLIP.

To enable removal of vector sequences from the left ITR, pAd5/L420-HSAwas partially digested with EcoRI and the linear fragment was isolated.An oligo of the sequence 5′ TTA AGT CGA C-3′ (SEQ ID NO: 11)corresponding to the incorporated '007 application was annealed toitself, resulting in a linker with a SalI site and EcoRI overhang. Thelinker was ligated to the partially digested pAd5/L420-HSA vector andclones were selected that had the linker inserted in the EcoRI site 23bp upstream of the left adenovirus ITR in pAd5/L420-HSA, resulting inpAd5/L420-HSA.sal.

To enable removal of vector sequences from the left ITR, pAd5/CLIP wasalso partially digested with EcoRI and the linear fragment was isolated.The EcoRI linker 5′ TTA AGT CGA C-3′ (SEQ ID NO: 12) corresponding tothe incorporated '007 application was ligated to the partially digestedpAd5/CLIP vector and clones were selected that had the linker insertedin the EcoRI site 23 bp upstream of the left adenovirus ITR, resultingin pAd5/CLIP.sal. The vector pAd5/L420-HSA was also modified to create aPacd site upstream of the left ITR. Hereto, pAd5/L420-HSA was digestedwith EcoRI and ligated to a Pacd linker (5′-AAT TGT CTT AAT TAA CCG CTTAA-3′ (SEQ ID NO:13) corresponding to the incorporated '007application). The ligation mixture was digested with Pacd and religatedafter isolation of the linear DNA from agarose gel to removeconcatamerized linkers. This resulted in adapter plasmidpAd5/L420-HSA.pac.

This plasmid was digested with AvrII and BglII. The vector fragment wasligated to a linker oligonucleotide digested with the same restrictionenzymes. The linker was made by annealing oligos of the followingsequence: PLL-1 (5′-GCC ATC CCT AGG AAG CTT GGT ACC GGT GAA TTC GCT AGCGTT AAC GGA TCC TCT AGA CGA GAT CTG G-3′ (SEQ ID NO:14) corresponding tothe incorporated '007 application) and PLL-2 (5′-CCA GAT CTC GTC TAG AGGATC CGT TAA CGC TAG CGA ATT CAC CGG TAC CAA GCT TCC TAG GGA TGG C-3′(SEQ ID NO:15) corresponding to the incorporated '007 application). Theannealed linkers were separately ligated to the AvrII/BglII-digestedpAd5/L420-HSA.pac fragment, resulting in pAdMire.pac. Subsequently, a0.7 kb ScaI/BsrGI fragment from pAd5/CLIP.sal containing the sal linkerwas cloned into the ScaI/BsrGI sites of the pAdMire.pac plasmid afterremoval of the fragment containing the pac linker. This resultingplasmid was named pAdMire.sal.

Plasmid pAd5/L420-HSA.pac was digested with AvrII and 5′ protruding endswere filled in using Klenow enzyme. A second digestion with HindIIIresulted in removal of the L420 promoter sequences. The vector fragmentwas isolated and ligated separately to a PCR fragment containing the CMVpromoter sequence. This PCR fragment was obtained after amplification ofCMV sequences from pCMVLacI (Stratagene) with the following primers:CMVplus (5′-GAT CGG TAC CAC TGC AGT GGT CAA TAT TGG CCA TTA GCC-3′ (SEQID NO:16) corresponding to the incorporated '007 application) andCMVminA (5′-GAT CAA GCT TCC AAT GCA CCG TTC CCG GC-3′ (SEQ ID NO:17)corresponding to the incorporated '007 application). The PCR fragmentwas first digested with PstI after which the 3′-protruding ends wereremoved by treatment with T4 DNA polymerase. Then the DNA was digestedwith HindIII and ligated into the AvrII/HindIII-digestedpAd5/L420-HSA.pac vector. The resulting plasmid was namedpAd5/CMV-HSA.pac. This plasmid was then digested with HindIII and BamHIand the vector fragment was isolated and ligated to the HindIII/BglIIpolylinker sequence obtained after digestion of pAdMire.pac. Theresulting plasmid was named pAdApt.pac and contains nucleotides −735 to+95 of the human CMV promoter/enhancer (M. Boshart et al., 1985).

The full length human EPO cDNA (Genbank accession number: MI 1319)containing a perfect Kozak sequence for proper translation was removedfrom the pUC18 backbone after a BamHI digestion. The cDNA insert waspurified over agarose gel and ligated into pAdApt.pac, which was alsodigested with BamHI, subsequently dephosphorylated at the 5′ and 3′insertion sites using SAP and also purified over agarose gel to removethe short BamHI-BamHI linker sequence. The obtained circular plasmid waschecked with KpnI, DdeI and NcoI restriction digestions that all gavethe right size bands. Furthermore, the orientation and sequence wasconfirmed by double stranded sequencing. The obtained plasmid with thehuman EPO cDNA in the correct orientation was named pAdApt.EPO (FIG. 6of the incorporated '007 application).

Example 3 Construction of UBS-54 Expression Vectors

The constant domains (CH1, -2 and -3) of the heavy chain of the humanimmunoglobulin G1 (IgG1) gene including intron sequences and connecting(“Hinge”) domain were generated by PCR using an upstream and a downstream primer. The sequence of the upstream primer (CAMH-UP) is 5′-GATCGA TAT CGC TAG CAC CAA GGG CCC ATC GGT C-3′ (SEQ ID NO: 18)corresponding to the incorporated '007 application, in which theannealing nucleotides are depicted in italics and two sequentialrestriction enzyme recognition sites (EcoRV and NheI) are underlined.

The sequence of the down stream primer (CAMH-DOWN) is: 5′-GAT CGT TTAAAC TCA TTT ACC CGG AGA CAG-3′ (SEQ ID NO:19) corresponding to theincorporated '007 application, in which the annealing nucleotides aredepicted in italics and the introduced PmeI restriction enzymerecognition site is underlined.

The order in which the domains of the human IgG1 heavy chain werearranged is as follows: CH1-intron-Hinge-intron-CH2-intron-CH3. The PCRwas performed on a plasmid (pCMgamma NEO Skappa Vgamma Cgamma hu)containing the heavy chain of a humanized antibody directed againstD-dimer from human fibrinogen (Vandamme et al., 1990). This antibody wasdesignated “15C5” and the humanization was performed with theintroduction of the human constant domains including intron sequences(Bulens et al., 1991). The PCR resulted in a product of 1621nucleotides. The NheI and PmeI sites were introduced for easy cloninginto the pcDNA2000/Hyg(−) polylinker. The NheI site encoded two aminoacids (Ala and Ser) that are part of the constant region CH1, but thatdid not hybridize to the DNA present in the template (Crowe et al.,1992).

The PCR product was digested with NheI and PmeI restriction enzymes,purified over agarose gel and ligated into a NheI and PmeI-digested andagarose gel-purified pcDNA2000/Hygro(−). This resulted in plasmidpHC2000/Hyg(−) (FIG. 7 of the incorporated '007 application), which canbe used for linking the human heavy chain constant domains, includingintrons to any possible variable region of any identified immunoglobulinheavy chain for humanization.

The constant domain of the light chain of the human immunoglobulin(IgG1) gene was generated by PCR using an upstream and a down streamprimer: The sequence of the upstream primer (CAML-UP) is 5′-GAT CCG TACGGT GGC TGCACCATC TGT C-3′ (SEQ ID NO:20) corresponding to theincorporated '007 application, in which the annealing nucleotides aredepicted in italics and an introduced SunI restriction enzymerecognition site is underlined.

The sequence of the down stream primer (CAML-DOWN) is 5′-GAT CGT TTA AACCTA ACA CTC TCC CCT GTT G-3′ (SEQ ID NO:21) corresponding to theincorporated '007 application, in which the annealing nucleotides are initalics and an introduced PmeI restriction enzyme recognition site isunderlined.

The PCR was performed on a plasmid (pCMkappa DHFR13 15C5 kappahumanized) carrying the murine signal sequence and murine variableregion of the light chain of 15C5 linked to the constant domain of thehuman IgG1 light chain (Vandamme et al., 1990; Bulens et al., 1991).

The PCR resulted in a product of 340 nucleotides. The SunI and PmeIsites were introduced for cloning into the pcDNA2001/DHFRwt polylinker.The SunI site encoded two amino acids (Arg and Thr) of which thethreonine residue is part of the constant region of human immunoglobulinlight chains, while the arginine residue is part of the variable regionof CAMPATH-1H (Crowe et al., 1992). This enabled subsequent 3′ cloninginto the SunI site, which was unique in the plasmid.

The PCR product was digested with SunI and PmeI restriction enzymespurified over agarose gel, ligated into a BamHI, PmeI-digested, andagarose gel-purified pcDNA2001/DHFRwt, which was blunted by Klenowenzyme and free nucleotides. Ligation in the correct orientationresulted in loss of the BamHI site at the 5′ end and preservation of theSunI and PmeI sites. The resulting plasmid was named pLC2001/DHFRwt(FIG. 8 of the incorporated '007 application), which plasmid can be usedfor linking the human light chain constant domain to any possiblevariable region of any identified immunoglobulin light chain forhumanization.

pNUT-C gamma (Huls et al., 1999) contains the constant domains, intronsand hinge region of the human IgG1 heavy chain (Huls et al., 1999) andreceived the variable domain upstream of the first constant domain. Thevariable domain of the gamma chain of fully humanized monoclonalantibody UBS-54 is preceded by the following leader peptide sequence:MACPGFLWALVISTCLEFSM (SEQ ID NO:22) corresponding to the incorporated'007 application (sequence: 5′-ATG GCA TGC CCT GGC TTC CTG TGG GCA CTTGTG ATC TCC ACC TGT CTT GAA TTT TCC ATG-3′) (SEQ ID NO:23) correspondingto the incorporated '007 application. This resulted in an insert ofapproximately 2 kb in length. The entire gamma chain was amplified byPCR using an upstream primer (UBS-UP) and the down stream primerCAMH-DOWN. The sequence of UBS-UP is as follows: 5′-GAT CAC GCG TGC TAGCCA CCA TGG CAT GCC CTG GCT TC-3′ (SEQ ID NO:24) corresponding to theincorporated '007 application in which the introduced MluI and NheIsites are underlined and the perfect Kozak sequence is italicized.

The resulting PCR product was digested with NheI and PmeI restrictionenzymes, purified over agarose gel and ligated to the pcDNA2000/Hygro(−)plasmid that is also digested with NheI and PmeI, dephosphorylated withtSAP and purified over gel. The resulting plasmid was namedpUBS-Heavy2000/Hyg(−) (FIG. 9 of the incorporated '007 application).pNUT-C kappa contains the constant domain of the light chain of humanIgG1 kappa (Huls et al., 1999) and received the variable domain of fullyhumanized monoclonal antibody UBS-54 kappa chain preceded by thefollowing leader peptide: MACPGFLWALVISTCLEFSM (SEQ ID NO:25)corresponding to the incorporated '007 application (sequence: 5′-ATG GCATGC CCT GGC TTC CTG TGG GCA CTT GTG ATC TCC ACC TGT CTT GAA TTT TCCATG-3′ (SEQ ID NO:26) corresponding to the incorporated '007application, for details on the plasmid see U-BiSys of Utrecht, NL).This resulted in an insert of approximately 1.2 kb in length.

The entire insert was amplified by PCR using the upstream primer UBS-UPand the down stream primer CAML-DOWN, hereby modifying the translationstart site. The resulting PCR product was digested with NheI and PmeIrestriction enzymes, purified over agarose gel and ligated topcDNA2001/DHFRwt that was also digested with NheI and PmeI,dephosphorylated by tSAP and purified over gel, resulting inpUBS-Light2001/DHFRwt (FIG. 10 of the incorporated '007 application). Toremove the extra intron which is located between the variable domain andthe first constant domain that is present in pNUT-Cgamma and to link thesignal peptide and the variable domain to the wild-type constant domainsof human IgG1 heavy chain, lacking a number of polymorphisms present inthe carboxy-terminal constant domain in pNUT-Cgamma, a PCR product isgenerated with primer UBS-UP and primer UBSHV-DOWN that has thefollowing sequence: 5′-GAT CGC TAG CTG TCGAGA CGG TGA CCA G-3′ (SEQ IDNO:27) corresponding to the incorporated '007 application, in which theintroduced NheI site is underlined and the annealing nucleotides areitalicized. The resulting PCR product is digested with NheI restrictionenzyme, purified over gel and ligated to a NheI-digested andSAP-dephosphorylated pHC2000/Hyg(−) plasmid that was purified over gel.The plasmid with the insert in the correct orientation and reading frameis named pUBS2-Heavy2000/Hyg(−) (FIG. 11 of the incorporated '007application).

For removal of an extra intron which is located between the variabledomain and the constant domain that is present in pNUT-Ckappa and tolink the signal peptide and the variable domain to the wild-typeconstant domain of human IgG1 light chain, a PCR product was generatedwith primer UBS-UP and primer UBSLV-DOWN that has the followingsequence: 5′-GAT CCG TAC GCT TGA TCT CCA CCT TGG TC -3′ (SEQ ID NO:28)corresponding to the incorporated '007 application, in which theintroduced SunI site is underlined and the annealing nucleotides are inbold. Then the resulting PCR product was digested with MluI and SunIrestriction enzymes, purified over gel and ligated to a MluI andSunI-digested pLC2001/DHFRwt plasmid that was purified over gel. Theresulting plasmid was named pUBS2-Light2001/DHFRwt (FIG. 12 of theincorporated '007 application).

The PCR product of the full-length heavy chain of UBS-54 is digestedwith NheI and PmeI restriction enzymes and blunted with Klenow enzyme.This fragment is ligated to the plasmid pcDNAs3000/DHFRwt that isdigested with BstXI restriction enzyme, blunted, dephosphorylated by SAPand purified over gel. The plasmid with the heavy chain insert is namedpUBS-Heavy3000/DHFRwt. Subsequently, the PCR of the light chain isdigested with MluI and PmeI restriction enzymes, blunted, purified overgel and ligated to pUBS-Heavy3000/DHFRwt that is digested with HpaI,dephosphorylated by tSAP and purified over gel. The resulting vector isnamed pUBS-3000/DHFRwt (FIG. 13 of the incorporated '007 application).The gene that encodes the heavy chain of UBS-54 without an intronbetween the variable domain and the first constant region and with awild-type carboxy terminal constant region (2031 nucleotides) ispurified over gel after digestion of pUBS2-2000/Hyg(−) with EcoRI andPmeI and treatment with Klenow enzyme and free nucleotides to blunt theEcoRI site. Subsequently, the insert is ligated to a pcDNAs3000/DHFRwtplasmid that is digested with BstXI, blunted, dephosphorylated with SAPand purified over gel. The resulting plasmid is namedpUBS2-Heavy3000/DHFRwt. pUBS2-Light2001/DHFRwt is then digested withEcoRV and PmeI, and the 755 nucleotide insert containing the signalpeptide linked to the variable domain of the kappa chain of UBS-54 andthe constant domain of human IgG1 kappa chain without an intron sequenceis purified over gel and ligated to pUBS2-Heavy3000/DHFRwt that isdigested with HpaI, dephosphorylated with tSAP and purified over gel.The resulting plasmid is named pUBS2-3000/DHFRwt (FIG. 14 of theincorporated '007 application).

Plasmid pRc/CMV (Invitrogen) was digested with BstBI restrictionenzymes, blunted with Klenow enzyme and subsequently digested with XmaIenzyme. The Neomycin resistance gene containing fragment was purifiedover agarose gel and ligated to pUBS-Light2001/DHFRwt plasmid that wasdigested with XmaI and PmlI restriction enzymes, followed bydephosphorylation with SAP and purified over gel to remove the DHFRcDNA. The resulting plasmid was named pUBS-Light2001/Neo(−). Thefragment was also ligated to a XmaI/PmlI-digested and gel-purifiedpcDNA2001/DHFRwt plasmid resulting in pcDNA2001/Neo. The PCR product ofthe UBS −54 variable domain and the digested and purified constantdomain PCR product were used in a three-point ligation with aMluI/PmeI-digested pcDNA2001/Neo. The resulting plasmid was namedpUBS2-Light2001/Neo.

Example 4 Construction of CAMPATH-1H Expression Vectors

Cambridge Bioscience Ltd. (UK) generates a 396 nucleotide fragmentcontaining a perfect Kozak sequence followed by the signal sequence andthe variable region of the published CAMPATH-1H light chain (Crowe etal., 1992). This fragment contains, on the 5′ end, an introduced andunique HindIII site and, on the 3′ end, an introduced and unique SunIsite and is cloned into an appropriate shuttle vector. This plasmid isdigested with HindIII and SunI and the resulting CAMPATH-1H light chainfragment is purified over gel and ligated into a HindIII/SunI-digestedand agarose gel-purified pLC2001/DHFRwt. The resulting plasmid is namedpCAMPATH-Light2001/DHFRwt. Cambridge Bioscience Ltd. (UK) generated a438 nucleotide fragment containing a perfect Kozak sequence followed bythe signal sequence and the published variable region of the CAMPATH-1Hheavy chain (Crowe et al., 1992), cloned into an appropriate cloningvector. This product contains a unique HindIII restriction enzymerecognition site on the 5′ end and a unique NheI restriction enzymerecognition site on the 3′ end. This plasmid was digested with HindIIIand NheI and the resulting CAMPATH-1H heavy chain fragment was purifiedover gel and ligated into a purified and HindIII/NheI-digestedpHC2000/Hyg(−). The resulting plasmid was namedpCAMPATH-Heavy2000/Hyg(−).

Example 5 Construction of 15C5 Expression Vectors

The heavy chain of the humanized version of the monoclonal antibody 15C5directed against human fibrin fragment D-dimer (Bulens et al., 1991;Vandamme et al., 1990) consisting of human constant domains includingintron sequences, hinge region and variable regions preceded by thesignal peptide from the 15C5 kappa light chain is amplified by PCR onplasmid “pCMgamma NEO Skappa Vgamma Cgamma hu” as a template usingCAMH-DOWN as a down stream primer and 15C5-UP as the upstream primer.15C5-UP has the following sequence: 5′-GA TCA CGC GTG CTA GCC ACC ATGGGT ACT CCT GCT CAG TTT CTT GGA ATC-3′ (SEQ ID NO:29) corresponding tothe incorporated '007 application, in which the introduced MluI and NheIrestriction recognition sites are underlined and the perfect Kozaksequence is italicized. To properly introduce an adequate Kozak context,the adenine at position +4 (the adenine in the ATG start codon is +1) isreplaced by a guanine, resulting in a mutation from an arginine into aglycine amino acid. To prevent primer dimerization, position +6 of theguanine is replaced by a thymine and the position +9 of the cytosine isreplaced by thymine. This latter mutation leaves the threonine residueintact. The resulting PCR was digested with NheI and PmeI restrictionenzymes, purified over gel and ligated to a NheI and PmeI-digestedpcDNA2000/Hygro(−), that is dephosphorylated by SAP and purified overagarose gel. The resulting plasmid is named p15C5-Heavy2000/Hyg(−). Thelight chain of the humanized version of the monoclonal antibody 15C5directed against human fibrin fragment D-dimer (Bulens et al., 1991;Vandamme et al., 1990) consisting of the human constant domain andvariable regions preceded by a 20 amino acid signal peptide is amplifiedby PCR on plasmid pCMkappa DHFR13 15C5kappa hu as a template, usingCAML-DOWN as a down stream primer and 15C5-UP as the upstream primer.The resulting PCR is digested with NheI and PmeI restriction enzymes,purified over gel and ligated to an NheI and PmeI-digestedpcDNA2001/DHFRwt that is dephosphorylated by SAP and purified overagarose gel. The resulting plasmid is named p15C5-Light2001/DHFRwt.

Example 6 Establishment of Methotrexate Hygromycin and G418 SelectionLevels

PER.C6 and PER.C6/E2A were seeded in different densities. The startingconcentration of methotrexate (MTX) in these sensitivity studies rangedbetween 0 nM and 2500 nM. The concentration which was just lethal forboth cell lines was determined; when cells were seeded in densities of100,000 cells per well in a six-well dish, wells were still 100%confluent at 10 nM and approximately 90 to 100% confluent at 25 nM,while most cells were killed at a concentration of 50 nM and above aftersix days to 15 days of incubation. These results are summarized in Table1 of the incorporated '007 application. PER.C6 cells were tested fortheir resistance to a combination of Hygromycin and G418 to selectoutgrowing stable colonies that expressed both heavy and light chainsfor the respective recombinant monoclonal antibodies encoded by plasmidscarrying either a hygromycin or a neomycin resistance gene. When cellswere grown on normal medium containing 100 μg/ml hygromycin and 250μg/ml G418, non-transfected cells were killed and stable colonies couldappear. (See, Example 7.)

CHO-dhfr cells ATCC deposit CRL9096 are seeded in different densities intheir respective culture medium. The starting concentration ofmethotrexate in these sensitivity studies ranges from approximately 0.5nM to 500 nM. The concentration, which is just lethal for the cell line,is determined and subsequently used directly after growth selection onhygromycin in the case of IgG heavy chain selection (hyg) and lightchain selection (dhfr).

Example 7 Transfection of EPO Expression Vectors to Obtain Stable CellLines

Cells of cell lines PER.C6 and PER.C6/E2A were seeded in 40-tissueculture dishes (10 cm diameter) with approximately 2 to 3 millioncells/dish and were kept overnight under their respective conditions(10% CO₂ concentration and temperature, which is 39° C. for PER.C6/E2Aand 37° C. for PER.C6). The next day, transfections were all performedat 37° C. using Lipofectamine (Gibco). After replacement with fresh(DMEM) medium after four hours, PER.C6/E2A cells were transferred to 39°C. again, while PER.C6 cells were kept at 37° C. Twenty dishes of eachcell line were transfected with 5 μg ScaI-digested pEPO2000/DHFRwt andtwenty dishes were transfected with 5 μg ScaI-digested pEPO2000/DHFRm,all according to standard protocols. Another 13 dishes served asnegative controls for methotrexate killing and transfection efficiency,which was approximately 50%. On the next day, MTX was added to thedishes in concentrations ranging between 100 and 1000 nM for DHFRwt and50,000 and 500,000 nM for DHFRm dissolved in medium containing dialyzedFBS. Cells were incubated over a period of four to five weeks. Tissuemedium (including MTX) was refreshed every two to three days. Cells weremonitored daily for death, comparing between positive and negativecontrols. Outgrowing colonies were picked and subcultured. No positiveclones could be subcultured from the transfectants that received themutant DHFR gene, most likely due to toxic effects of the highconcentrations of MTX that were applied. From the PER.C6 and PER.C6/E2Acells that were transfected with the wild-type DHFR gene, only celllines could be established in the first passages when cells were grownon 100 nM MTX, although colonies appeared on dishes with 250 and 500 nMMTX. These clones were not viable during subculturing, and werediscarded.

Example 8 Sub-Culturing of Transfected Cells

From each cell line, approximately 50 selected colonies that wereresistant to the threshold MTX concentration were grown subsequently in96-well, 24-well, and 6-well plates and T25 flasks in their respectivemedium plus MTX. When cells reached growth in T25 tissue culture flasks,at least one vial of each clone was frozen and stored, and wassubsequently tested for human recombinant EPO production. For this, thecommercial ELISA kit from R&D Systems was used (Quantikine IVD humanEPO, Quantitative Colorimetric Sandwich ELISA, cat.# DEPOO). Since thedifferent clones appeared to have different growth characteristics andgrowth curves, a standard for EPO production was set as follows: At day0, cells were seeded in T25 tissue culture flasks in concentrationsranging between 0.5 to 1.5 million per flask. At day 4, supernatant wastaken and used in the EPO ELISA. From this, the production level was setas ELISA units per million seeded cells per day. (U/1E6/day) A number ofthese clones are given in Table 2 of the incorporated '007 patentapplication.

The following selection of good producer clones was based on highexpression, culturing behavior and viability. To allow checks forlong-term viability, suspension growth in roller bottles and bioreactorduring extended time periods, more vials of the best producer cloneswere frozen, and the following best producers of each cell line wereselected for further investigations P8, P9, E17 and E55 in which “P”stands for PER.C6 and “E” stands for PER.C6/E2A. These clones aresubcultured and subjected to increasing doses of methotrexate in a timespan of two months. The concentration starts at the thresholdconcentration and increases to approximately 0.2 mM. During these twomonths, EPO ELISA experiments are performed on a regular basis to detectan increase in EPO production. At the highest methotrexateconcentration, the best stable producer is selected and compared to theamounts from the best CHO clone and used for cell banking (RL). Fromevery other clone, five vials are frozen. The number of amplified EPOcDNA copies is detected by Southern blotting.

Example 9 EPO Production in Bioreactors

The best performing EPO producing transfected stable cell line ofPER.C6, P9, was brought into suspension and scaled up to 1 to 2 literfermentors. To get P9 into suspension, attached cells were washed withPBS and subsequently incubated with JRH ExCell 525 medium for PER.C6(JRH Biosciences), after which the cells loosen from the flask and formthe suspension culture. Cells were kept at two concentrations of MTX: 0nM and 100 nM. General production levels of EPO that were reached atthese concentrations (in roller bottles) were respectively 1500 and 5700units per million seeded cells per day. Although the lower yields in theabsence of MTX can be explained by removal of the integrated DNA, itseems as if there is a shut-down effect of the integrated DNA sincecells that are kept at lower concentrations of MTX for longer periods oftime are able to reach their former yields when they are transferred to100 nM MTX concentrations again. (See, Example 11.)

Suspension P9 cells were grown normally with 100 nM MTX and used forinoculation of bioreactors. Two bioreactor settings were tested:perfusion and repeated batch cultures.

A. Perfusion in a 2 Liter Bioreactor.

Cells were seeded at a concentration of 0.5×10⁶ cells per ml andperfusion was started at day 3 after cells reached a density ofapproximately 2.3×10⁶ cells per ml. The perfusion rate was 1 volume per24 hours with a bleed of approximately 250 ml per 24 hours. In thissetting, P9 cells stayed at a constant density of approximately 5×10⁶cells per ml and a viability of almost 95% for over a month. The EPOconcentration was determined on a regular basis and is shown in FIG. 15(of the incorporated '007 application). In the 2 liter perfusedbioreactor the P9 cells were able to maintain a production level ofapproximately 6000 ELISA units per ml. With a perfusion rate of oneworking volume per day (1.5 to 1.6 liter), this means that in this 2liter setting, the P9 cells produced approximately 1×10⁷ units per dayper 2 liter bioreactor in the absence of MTX.

B. Repeated Batch in a 2 Liter Bioreactor.

P9 suspension cells that were grown on roller bottles were used toinoculate a 2 liter bioreactor in the absence of MTX and were left togrow until a density of approximately 1.5 million cells per ml, afterwhich a third of the population was removed (+/−1 liter per 2 to 3 days)and the remaining culture was diluted with fresh medium to reach again adensity of 0.5 million cells per ml. This procedure was repeated forthree weeks and the working volume was kept at 1.6 liter. EPOconcentrations in the removed medium were determined and shown in FIG.16 of the incorporated '007 application. The average concentration wasapproximately 3000 ELISA units per ml. With an average period of twodays after which the population was diluted, this means that, in this 2liter setting, the P9 cells produced approximately 1.5×10⁶ units per dayin the absence of MTX.

C. Repeated Batch in a 1 Liter Bioreactor with Different Concentrationsof Dissolved Oxygen, Temperatures and pH Settings.

Fresh P9 suspension cells were grown in the presence of 100 nM MTX inroller bottles and used for inoculation of 4×1 liter bioreactors to adensity of 0.3 million cells per ml in JRH ExCell 525 medium. EPO yieldswere determined after 3, 5 and 7 days. The first settings that weretested were: 0.5%, 10%, 150% and as a positive control 50% DissolvedOxygen (% DO). 50% DO is the condition in which PER.C6 and P9 cells arenormally kept. In another run, P9 cells were inoculated and tested forEPO production at different temperatures (32° C., 34° C., 37° C. and 39°C.) in which 37° C. is the normal setting for PER.C6 and P9, and in thethird run, fresh P9 cells were inoculated and tested for EPO productionat different pH settings (pH 6.5, pH 6.8, pH 7.0 and pH 7.3). PER.C6cells are normally kept at pH 7.3. An overview of the EPO yields (threedays after seeding) is shown in FIG. 17 of the incorporated '007application. Apparently, EPO concentrations increase when thetemperature is rising from 32 to 39° C. as was also seen with PER.C6/E2Acells grown at 39° C. (Table 4) (of the incorporated '007 application),and 50% DO is optimal for P9 in the range that was tested here. At pH6.5, cells cannot survive since the viability in this bioreactor droppedbeneath 80% after seven days. EPO samples produced in these settings arechecked for glycosylation and charge in 2D electrophoresis. (See, alsoExample 17.)

Example 10 Amplification of the DHFR Gene

A number of cell lines described in Example 8 were used in anamplification experiment to determine the possibility of increasing thenumber of DHFR genes by increasing the concentration of MTX in a timespan of more than two months. The concentration started at the thresholdconcentration (100 nM) and increased to 1800 nM with in-between steps of200 nM, 400 nM, 800 nM and 1200 nM. During this period, EPO ELISAexperiments were performed on a regular basis to detect the units permillion seeded cells per day (FIG. 18 of the incorporated '007application). At the highest MTX concentration (1800 nM), some vialswere frozen. Cell pellets were obtained and DNA was extracted andsubsequently digested with BglII, since this enzyme cuts around thewild-type DHFR gene in pEPO200/DHFRwt (FIG. 5 of the incorporated '007application), so a distinct DHFR band of that size would bedistinguishable from the endogenous DHFR bands in a Southern blot. ThisDNA was run and blotted and the blot was hybridized with a radioactiveDHFR probe and subsequently with an adenovirus E1 probe as a backgroundcontrol (FIG. 19 of the incorporated '007 application). The intensitiesof the hybridizing bands were measured in a phosphorimager and correctedfor background levels. These results are shown in Table 3 of theincorporated '007 application. Apparently, it is possible to obtainamplification of the DHFR gene in PER.C6 cells, albeit in this case onlywith the endogenous DHFR and not with the integrated vector.

Example 11 Stability of EPO Expression in Stable Cell Lines

A number of cell lines mentioned in Example 8 were subject to long termculturing in the presence and absence of MTX. EPO concentrations weremeasured regularly in which 1.0 to 1.5×10⁶ cells per T25 flask wereseeded and left for four days to calculate the production levels of EPOper million seeded cells per day. The results are shown in FIG. 20 ofthe incorporated '007 application. From this, it is concluded that thereis a relatively stable expression of EPO in P9 cells when cells arecultured in the presence of MTX and that there is a decrease in EPOproduction in the absence of MTX. However, when P9 cells were placed on100 nM MTX again after being cultured for a longer period of timewithout MTX, the expressed EPO reached its original level (+/−3000 ELISAunits per million seeded cells per day), suggesting that the integratedplasmids are shut off but are stably integrated and can be switched backon again. It seems as if there are differences between the cell lines P8and P9 because the production level of P8 in the presence of MTX isdecreasing in time over a high number of passages (FIG. 20A of theincorporated '007 application), while P9 production is stable for atleast 62 passages (FIG. 20B of the incorporated '007 application).

Example 12 Transient Expression of Recombinant EPO on Attached andSuspension Cells after Plasmid DNA Transfections

pEPO2000/DHFRwt, pEPO2000/DHFRm and pAdApt.EPO plasmids from Example 2are purified from E. coli over columns, and are transfected usinglipofectamine, electroporation, PEI or other methods. PER.C6 orPER.C6/E2A cells are counted and seeded in DMEM plus serum or JRH ExCell525 medium or the appropriate medium for transfection in suspension.Transfection is performed at 37° C. up to 16 hours, depending on thetransfection method used, according to procedures known by a personskilled in the art. Subsequently, the cells are placed at differenttemperatures and the medium is replaced by fresh medium with or withoutserum. In the case when it is necessary to obtain medium that completelylacks serum components, the fresh medium lacking serum is removed againafter 3 hours and replaced again by medium lacking serum components. Fordetermination of recombinant EPO production, samples are taken atdifferent time points. Yields of recombinant protein are determinedusing an ELISA kit (R&D Systems) in which 1 Unit equals approximately 10ng of recombinant CHO-produced EPO protein (100,000 Units/mg). The cellsused in these experiments grow at different rates, due to their origin,characteristics and temperature. Therefore, the amount of recombinantEPO produced is generally calculated in ELISA units/10⁶ seededcells/day, taking into account that the antisera used in the ELISA kitdo not discriminate between non- and highly glycosylated recombinantEPO. Generally, samples for these calculations are taken at day 4 afterreplacing the medium upon transfection.

PER.C6/E2A cells, transfected at 37° C. using lipofectamine andsubsequently grown at 39° C. in the presence of serum, typicallyproduced 3100 units/10⁶ cells/day. In the absence of serum componentswithout any refreshment of medium lacking serum, theselipofectamine-transfected cells typically produced 2600 units/10⁶cells/day. PER.C6 cells, transfected at 37° C. using lipofectamine andsubsequently grown at 37° C. in the presence of serum, typicallyproduced 750 units/10⁶ cells/day and, in the absence of serum, 590units/10⁶ cells/day. For comparison, the same expression plasmidspEPO2000/DHFRwt and pEPO2000/DHFRm were also applied to transfect CHOcells (ECACC deposit no. 85050302) using lipofectamine, PEI, calciumphosphate procedures and other methods. When CHO cells were transfectedusing lipofectamine and subsequently cultured in Hams F12 medium in thepresence of serum, a yield of 190 units/10⁶ cells/day was obtained. Inthe absence of serum, 90 units/10⁶ cells/day were produced, althoughhigher yields can be obtained when transfections are being performed inDMEM.

Different plates containing attached PER.C6/E2A cells were alsotransfected at 37° C. with pEPO2000/DHFRwt plasmid and subsequentlyplaced at 32° C., 34° C., 37° C. or 39° C. to determine the influence oftemperature on recombinant EPO production. A temperature-dependentproduction level was observed ranging from 250 to 610 units/10⁶ seededcells/day, calculated from a day 4 sample, suggesting that thedifference between production levels observed in PER.C6 and PER.C6/E2Ais partly due to incubation temperatures (see, also FIG. 17 of theincorporated '007 application). Since PER.C6/E2A grows well at 37° C.,further studies were performed at 37° C.

Different plates containing attached PER.C6 and PER.C6/E2A cells weretransfected with pEPO2000/DHFRwt, pEPO2000/DHFRm and pAdApt.EPO usinglipofectamine. Four hours after transfection, the DMEM was replaced witheither DMEM plus serum or JRH medium lacking serum and EPO was allowedto accumulate in the supernatant for several days to determine theconcentrations that are produced in the different mediums. PER.C6 cellswere incubated at 37° C., while PER.C6/E2A cells were kept at 39° C.Data from the different plasmids were averaged since they contain asimilar expression cassette. Calculated from a day 6 sample, thefollowing data were obtained: PER.C6 grown in DMEM produced 400units/10⁶ seeded cells/day, and when they were kept in JRH medium, theyproduced 300 units/10⁶ seeded cells/day. PER.C6/E2A grown in DMEMproduced 1800 units/10⁶ seeded cells/day, and when they were kept inJRH, they produced 1100 units/10⁶ seeded cells/day. Again, a cleardifference was observed in production levels between PER.C6 andPER.C6/E2A, although this might partly be due to temperaturedifferences. There was, however, a significant difference withPER.C6/E2A cells between the concentration in DMEM vs. the concentrationin JRH medium, although this effect was almost completely lost in PER.C6cells.

EPO expression data obtained in this system are summarized in Table 4(of the incorporated '007 application). PER.C6 and derivatives thereofcan be used for scaling up the DNA transfections system. According toWurm and Bernard (1999), transfections on suspension cells can beperformed at 1 to 10 liter set-ups in which yields of 1 to 10 mg/l (0.1to 1 pg/cell/day) of recombinant protein have been obtained usingelectroporation. A need exists for a system in which this can be wellcontrolled and yields might be higher, especially for screening of largenumbers of proteins and toxic proteins that cannot be produced in astable setting. With the lipofectamine transfections on the best PER.C6cells in the absence of serum, we reached 590 units/million cells/day(+/−5.9 pg/cell/day when 1 ELISA unit is approximately 10 ng EPO), whilePER.C6/E2A reached 31 pg/cell/day (in the presence of serum). The mediumused for suspension cultures of PER.C6 and PER.C6/E2A (JRH ExCell 525)does not support efficient transient DNA transfections using componentslike PEI. Therefore, the medium is adjusted to enable production ofrecombinant EPO after transfection of pEPO2000/DHFRwt and pEPO2000/DHFRmcontaining a recombinant human EPO cDNA, and pcDNA2000/DHFRwt containingother cDNAs encoding recombinant proteins.

One to 10 liter suspension cultures of PER.C6 and PER.C6/E2A growing inadjusted medium to support transient DNA transfections using purifiedplasmid DNA are used for electroporation or other methods, performingtransfection with the same expression plasmids. After several hours, thetransfection medium is removed and replaced by fresh medium withoutserum. The recombinant protein is allowed to accumulate in thesupernatant for several days, after which the supernatant is harvestedand all the cells are removed. The supernatant is used for down streamprocessing to purify the recombinant protein.

Example 13 Generation of AdApt.EPO Recombinant Adenoviruses

pAdApt.EPO was co-transfected with the pWE/Ad.Af1II-rITR.tetO-E4,pWE/Ad.Af1II-rITR.DE2A, and pWE/Ad.Af1II-rITR.DE2A.tetO-E4 cosmids inthe appropriate cell lines using procedures known to persons skilled inthe art. Subsequently, cells were left at their appropriate temperaturesfor several days until full cytopathic effect (“CPE”) was observed. Thencells were applied to several freeze/thaw steps to free all viruses fromthe cells, after which the cell debris was spun down. ForIG.Ad5/AdApt.EPO.dE2A, the supernatant was used to infect cells,followed by an agarose overlay for plaque purification using severaldilutions. After a number of days, when single plaques were clearlyvisible in the highest dilutions, nine plaques and one negative control(picked cells between clear plaques, so most likely not containingvirus) were picked and checked for EPO production on A549 cells. Allplaque picked viruses were positive and the negative control did notproduce recombinant EPO. One positive producer was used to infect theappropriate cells and to propagate virus starting from a T-25 flask to aroller bottle setting. Supernatants from the roller bottles were used topurify the virus, after which the number of virus particles (vps) wasdetermined and compared to the number of infectious units (IUs) usingprocedures known to persons skilled in the art. Then, the vp/IU ratiowas determined.

Example 14 Infection of Attached and Suspension PER.C6 Cells withIG.Ad5/AdApt.EPO.dE2A

Purified viruses from Example 13 were used for transient expression ofrecombinant EPO in PER.C6 cells and derivatives thereof.IG.Ad5/AdApt.EPO.dE2A virus was used to infect PER.C6 cells, whileIG.Ad5/AdApt.EPO.tetOE4 and IG.Ad5/AdApt.EPO.dE2A.tetOE4 viruses can beused to infect PER.C6/E2A cells to lower the possibility of replicationand, moreover, to prevent inhibition of recombinant protein productiondue to replication processes. Infections were performed on attachedcells as well as on suspension cells in their appropriate medium usingranges of multiplicities of infection (moi's): 20, 200, 2000, 20000vp/cell. Infections were performed for four hours in different settingsranging from six-well plates to roller bottles, after which the viruscontaining supernatant was removed. The cells were washed with PBS ordirectly refreshed with new medium. Then, cells were allowed to producerecombinant EPO for several days, during which samples were taken andEPO yields were determined. Also, the number of viable cells compared todead cells was checked. The amount of EPO that was produced was againcalculated as ELISA unit seeded cells/day, because the different celllines have different growth characteristics due to their passage numberand environmental circumstances such as temperature and selectivepressures. Suspension growing PER.C6 cells were seeded in 250 ml JRHExCell 525 medium in roller bottles (1 million cells per ml) andsubsequently infected with purified IG.Ad5/AdApt.EPO.dE2A virus with amoi of 200 vp/cell. The estimation used for vp determination was high(vp/tU ratio of this batch is 330, which indicates an moi of 0.61IUs/cell). Thus, not all cells were hit by an infectious virus. Atypical production of recombinant EPO in this setting from a day 6sample was 190 units/10⁶ seeded cells/day, while in a setting in which50% of the medium including viable and dead cells was replaced by freshmedium, approximately 240 units/10⁶ cells/day were obtained. Therefreshment did not influence the viability of the viable cells, but theremoved recombinant protein could be added to the amount that wasobtained at the end of the experiment, albeit present in a largervolume. An identical experiment was performed with the exception thatcells were infected with a moi of 20 vp/cell, resembling approximately0.06 Infectious Units/cell. Without refreshment, a yield of 70 ELISAunits/10⁶ cells/day was obtained, while in the experiment in which 50%of the medium was refreshed at day 3, a typical amount of 80 units/10⁶cells/day was measured. This indicates that there is a dose responseeffect when an increasing number of infectious units are used forinfection of PER.C6 cells.

Furthermore, PER.C6 cells growing in DMEM were seeded in six-well platesand left overnight in 2 ml DMEM with serum to attach. The next day,cells were infected with another batch of IG.Ad5/AdApt.EPO.dE2A virus(vp/IU ratio 560) with a moi of 200 vp/cells (0.35 InfectiousUnits/cell). After four hours, the virus containing medium was removedand replaced by fresh medium including serum, and cells were left toproduce recombinant EPO for more than two weeks with replacement of themedium with fresh medium every day. The yield of recombinant EPOproduction calculated from a day 4 sample was 60 units/10⁶ cells/day.

Expression data obtained in this system have been summarized in Table 5(of the incorporated '007 application).

Due to the fact that a tTA-tetO regulated expression of the Early region4 of adenovirus (E4) impairs the replication capacity of the recombinantvirus in the absence of active E4, it is also possible to use thepossible protein production potential of the PER.C6/E2A as well as itsparental cell line PER.C6 to produce recombinant proteins in a settingin which a recombinant adenovirus is carrying the human EPO cDNA as thetransgene and in which the E4 gene is under the control of a tet operon.Then, very low levels of E4 mRNA are being produced, resulting in verylow but detectable levels of recombinant and replicating virus in thecell line PER.C6/E2A and no detectable levels of this virus in PER.C6cells. To produce recombinant EPO in this way, the two virusesIG.Ad5/AdApt.EPO.tetOE4 and IG.Ad5/AdApt.EPO.dE2A.tetOE4 are used toinfect PER.C6 cells and derivatives thereof. Attached and suspensioncells are infected with different moi's of the purified adenoviruses insmall settings (six-well plates and T25 flasks) and large settings(roller bottles and fermentors). Samples are taken at different timepoints and EPO levels are determined.

Since viruses that are deleted in E1 and E2A in the viral backbone canbe complemented in PER.C6/E2A cells but not in the original PER.C6cells, settings are used in which a mixture of both cell lines iscultured in the presence of IG.Ad5/AdApt.EPO.dE2A virus. The virus willreplicate in PER.C6/E2A, followed by lysis of the infected cells and asubsequent infection of PER.C6 or PER.C6/E2A cells. In contrast, inPER.C6 cells, the virus will not replicate and the cells will not lysedue to viral particle production, but will produce recombinant EPO thatwill be secreted in the supernatant. A steady stateculture/replication/EPO production system is set up in which freshmedium and fresh PER.C6 and PER.C6/E2A cells are added at a constantflow, while used medium, dead cells and debris are removed. Togetherwith this, recombinant EPO is taken from the system and used forpurification in a down stream processing procedure in which virusparticles are removed.

Example 15 Purification and Analysis of Recombinant EPO

Large batches of growing cells are produced in bioreactors; the secretedrecombinant human EPO protein is purified according to procedures knownby one of skill in the art. The purified recombinant human EPO proteinfrom PER.C6 and PER.C6/E2A stable clones or transfectants is checked forglycosylation and folding by comparison with commercially available EPOand EPO purified from human origin (urine) using methods known to one ofskill in the art (see, Examples 16 and 17). Purified and glycosylatedEPO proteins from PER.C6 and PER.C6/E2A are tested for biologicalactivity in in vitro experiments and in mouse spleens as described(Krystal (1983) and in vitro assays (see, Example 18).

Example 16 Activity of Beta-galactoside Alpha 2,6-sialyltransferase inPER.C6

It is known that CHO cells do not contain a gene for beta-galactosidealpha 2,6-sialyltransferase, resulting in the absence of alpha2,6-linked sialic acids at the terminal ends of—and O-linkedoligosaccharides of endogenous and recombinant glycoproteins produced onthese CHO cells. Since the alpha 2,3-sialyltransferase gene is presentin CHO cells, proteins that are produced on these cells are typicallyfrom the 2,3 linkage type. EPO that was purified from human urine does,however, contain both alpha 2,3- and alpha 2,6-linked sialic acids. Todetermine whether PER.C6 cells, being a human cell line, are able toproduce recombinant EPO containing both alpha 2,3- and alpha2,6-linkages, a direct neuraminidase assay was performed on recombinantEPO produced on PER.C6 cells after transfection with EPO expressionvectors. As a control, commercially available Eprex samples were used,which were derived from CHO cells and which should only contain sialicacid linkages of the alpha 2,3 type. The neuraminidases that were usedwere from Newcastle Disease Virus (NDV) that specifically cleaves alpha2,3-linked neuraminic acids (sialic acids) from—and O-linked glycans,and from Vibrocholerae (VC) that non-specifically cleaves allterminal—or O-linked sialic acids (alpha 2,3, alpha 2,6 and alpha 2,8linkages). Both neuraminidases were from Boehringer and were incubatedwith the samples according to guidelines provided by the manufacturer.Results are shown in FIG. 21A (of the incorporated '007 application). Inlanes 2 and 3 (treatment with NDV neuraminidase), a slight shift isobserved as compared to lane 1 (non-treated PER.C6 EPO). When this EPOsample was incubated with VC derived neuraminidase, an even fastermigrating band is observed as compared to NDV treated samples. However,with the commercially available Eprex, only a shift was observed whenNDV derived neuraminidase was applied (lanes 6 and 7 compared to thenon-treated sample in lane 5) and not when VC neuraminidase was used(lane 8).

To definitely establish that no sialic acids of the alpha 2,6 linkagetype are present on CHO cells, but that they do exist in proteinspresent on the cell surface of PER.C6 cells, the following experimentwas performed: CHO cells were released from the solid support usingtrypsin-EDTA, while for PER.C6, suspension cells were used. Bothsuspensions were washed once with Mem-5% FBS and incubated in thismedium for one hour at 37° C. After washing with PBS, the cells wereresuspended to approximately 10⁶ cells/ml in binding medium(Tris-buffered saline, pH 7.5, 0.5% BSA, and 1 mM each of MgCl₂, MnCl₂and CaCl₂). Aliquots of the cells were incubated for 1 hour at roomtemperature with DIG-labeled lectins, Sambucus nigra agglutinin (“SNA”)and Maackia amurensis agglutinin (“MAA”), which specifically bind tosialic acid linkages of the alpha 2,6 Gal and alpha 2,3 Gal types,respectively. Control cells were incubated without lectins. After onehour, both lectin-treated and control cells were washed with PBS andthen incubated for one hour at room temperature with FITC-labeledanti-DIG antibody (Boehringer-Mannheim). Subsequently, the cells werewashed with PBS and analyzed for fluorescence intensity on a FACsortapparatus (Becton Dickinson). The FACS analysis is shown in FIG. 21B (ofthe incorporated '007 application). When the SNA lectin is incubatedwith CHO cells, no shift is seen as compared to non-treated cells, whilewhen this lectin is incubated with PER.C6 cells, a clear shift (darkfields) is observed as compared to non-treated cells (open fields). Whenboth cell lines are incubated with the MAA lectin, both cell lines givea clear shift as compared to non-treated cells.

From these EPO digestions and FACS results, it is concluded that thereis a beta-galactoside alpha 2,6 sialyltransferase activity present inhuman PER.C6 cells which is absent in CHO cells.

Example 17 Determination of Sialic Acid Content in PER.C6 Produced EPO

The terminal neuraminic acids (or sialic acids) that are present onthe—and O-linked glycans of EPO protect the protein from clearance fromthe bloodstream by enzymes in the liver. Moreover, since these sialicacids are negatively charged, one can distinguish between different EPOforms depending on their charge or specific pI. Therefore, EPO producedon PER.C6 and CHO cells was used in two-dimensional electrophoresis inwhich the first dimension separates the protein on charge (pH range3-10) and the second dimension separates the proteins further onmolecular weight. Subsequently, the proteins were blotted and detectedin a western blot with an anti-EPO antibody.

It is also possible to detect the separated EPO protein by staining thegel using Coomassie blue or silver staining methods, subsequentlyremoving different spots from the gel and determining the specificglycan composition of the different—or O-linked glycosylations that arepresent on the protein by mass spectrometry.

In FIG. 22A of the incorporated '007 application, a number of EPOsamples are shown that were derived from P9 supernatants. P9 is thePER.C6 cell line that stably expresses recombinant human EPO (see,Example 8). These samples were compared to commercially available Eprex,which contains only EPO forms harboring approximately 9 to 14 sialicacids. Eprex should, therefore, be negatively charged and be focusingtowards the pH3 side of the gel. FIG. 22B (of the incorporated '007application) shows a comparison between EPO derived from P9 in anattached setting in which the cells were cultured on DMEM medium and EPOderived from CHO cells that were transiently transfected with thepEPO2000/DHFRwt vector. Apparently, the lower forms of EPO cannot bedetected in the CHO samples, whereas all forms can be seen in the P9sample. The sialic acid content is given by numbering the bands thatwere separated in the first dimension from 1 to 14. It is not possibleto determine the percentage of each form of EPO molecules present in themixtures because the western blot was performed using ECL, and becauseit is unknown whether glycosylation of the EPO molecule or transfer ofthe EPO molecule to the nitrocellulose inhibits recognition of the EPOmolecule by the antibody. However, it is possible to determine thepresence of the separate forms of sialic acid containing EPO molecules.It can be concluded that PER.C6 is able to produce the entire range of14 sialic acid containing isoforms of recombinant human EPO.

Example 18 In Vitro Functionality of PER.C6 Produced EPO

The function of recombinant EPO in vivo is determined by its half-lifein the bloodstream. Removal of EPO takes place by liver enzymes thatbind to galactose residues in the glycans that are not protected bysialic acids and by removal through the kidney. Whether this filteringby the kidney is due to misfolding or due to under- or mis-glycosylationis unknown. Furthermore, EPO molecules that reach their targets in thebone marrow and bind to the EPO receptor on progenitor cells are alsoremoved from circulation. Binding to the EPO receptor and down streamsignaling depends heavily on a proper glycosylation status of the EPOmolecule. Sialic acids can, to some extent, inhibit binding of EPO tothe EPO receptor, resulting in a lower effectiveness of the protein.However, since the sialic acids prevent EPO from removal, these sugarsare essential for its function to protect the protein on its travel tothe EPO receptor. When sialic acids are removed from EPO in vitro, abetter binding to the receptor occurs, resulting in a stronger downstream signaling. This means that the functionalities in vivo and invitro are significantly different, although a proper EPO receptorbinding property can be checked in vitro despite the possibility of anunder-sialylation causing a short half-life in vivo (Takeuchi et al.,1989).

Several in vitro assays for EPO functionality have been described ofwhich the stimulation of the IL3, GM-CSF and EPO-dependent human cellline TF-1 is most commonly used. Hereby, one can determine the number ofin vitro units per mg (Kitamura et al., 1989; Hammerling et al., 1996).Other in vitro assays are the formation of red colonies under an agaroselayer of bone marrow cells that were stimulated to differentiate by EPO,the incorporation of 59Fe into heme in cultured mouse bone marrow cells(Krystal et al., 1981 and 1983; Takeuchi et al., 1989), in rat bonemarrow cells (Goldwasser et al., 1975) and the Radio Immuno Assay (RIA)in which the recognition of EPO for antisera is determined.

EPO produced on PER.C6/E2A cells was used to stimulate TF-1 cells asfollows: Cells were seeded in 96-well plates with a density of around10,000 cells per well in medium lacking IL3 or GM-CSF, which are thegrowth factors that can stimulate indefinite growth of these cells inculture. Subsequently, medium is added, resulting in finalconcentrations of 0.0001, 0.001, 0.01, 0.1, 1 and 10 units per ml. Theseunits were determined by ELISA, while the units of the positive controlEprex were known (4000 units per ml) and were diluted to the sameconcentration. Cells were incubated with these EPO samples for fourdays, after which an MTS assay (Promega) was performed to check forviable cells by fluorescence measurement at 490 nm (fluorescence isdetectable after transfer of MTS into formazan). FIG. 23 of theincorporated '007 application shows the activity of two samples derivedfrom PER.C6/E2A cells that were transfected with an EPO expressionvector and subsequently incubated at 37° C. and 39° C. for four days.The results suggest that samples obtained at 39° C. are more active thansamples obtained at 37° C., which might indicate that the sialic acidcontent is suboptimal at higher temperatures. It is hereby shown thatPER.C6 produced EPO can stimulate TF-1 cells in an in vitro assay,strongly suggesting that the EPO that is produced on this human cellline can interact with the EPO receptor and stimulate differentiation.

Example 19 Production of Recombinant Murine, Humanized and HumanMonoclonal Antibodies in PER.C6 and PER.C6/E2A

A. Transient DNA Transfections

cDNAs encoding heavy and light chains of murine, humanized and humanmonoclonal antibodies (mAbs) are cloned in two different systems: one inwhich the heavy and light chains are integrated into one single plasmid(a modified pcDNA2000/DHFRwt plasmid) and the other in which heavy andlight chain cDNAs are cloned separately into two different plasmids(see, Examples 1, 3, 4 and 5). These plasmids can carry the sameselection marker (like DHFR) or they carry their own selection marker(one that contains the DHFR gene and one that contains, for instance,the neo-resistance marker). For transient expression systems, it doesnot matter what selection markers are present in the backbone of thevector since no subsequent selection is being performed. In the commonand regular transfection methods used in the art, equal amounts ofplasmids are transfected. A disadvantage of integrating both heavy andlight chains on one single plasmid is that the promoters that aredriving the expression of both cDNAs might influence each other,resulting in non-equal expression levels of both subunits, although thenumber of cDNA copies of each gene is exactly the same.

Plasmids containing the cDNAs of the heavy and light chain of a murineand a humanized monoclonal antibody are transfected and, after severaldays, the concentration of correctly folded antibody is determined usingmethods known to persons skilled in the art. Conditions such astemperature and used medium are checked for both PER.C6 and PER.C6/E2Acells. Functionality of the produced recombinant antibody is controlledby determination of affinity for the specified antigen.

B. Transient Viral Infections

cDNAs encoding a heavy and a light chain are cloned in two differentsystems: one in which the heavy and light chains are integrated into onesingle adapter plasmid (a modified pAdApt.pac) and the other in whichheavy and light chain cDNAs are cloned separately into two differentadapters (each separately in pAdApt.pac). In the first system, virusesare propagated that carry an E1 deletion (dE1) together with an E2Adeletion (dE2A) or both deletions in the context of a tetOE4 insertionin the adenoviral backbone. In the second system, the heavy and lightchains are cloned separately in pAdApt.pac and separately propagated toviruses with the same adenoviral backbone. These viruses are used toperform single or co-infections on attached and suspension growingPER.C6 and PER.C6/E2A cells. After several days, samples are taken todetermine the concentration of full length recombinant antibodies, afterwhich the functionality of these antibodies is determined using thespecified antigen in affinity studies.

C. Stable Production and Amplification of the Integrated Plasmid.

Expression plasmids carrying the heavy and light chain together andplasmids carrying the heavy and light chain separately are used totransfect attached PER.C6 and PER.C6/E2A and CHO-dhfr cells.Subsequently, cells are exposed to MTX and/or hygromycin and neomycin toselect for integration of the different plasmids. Moreover, a doubleselection with G418 and hygromycin is performed to select forintegration of plasmids that carry the neomycin and hygromycinresistance gene. Expression of functional full length monoclonalantibodies is determined and best expressing clones are used forsubsequent studies including stability of integration, copy numberdetection, determination of levels of both subunits and ability toamplify upon increase of MTX concentration after the best performingcell lines are used for mAb production in larger settings such asperfused and (fed-) batch bioreactors, after which optimization ofquantity and quality of the mAbs is executed.

Example 20 Transfection of mAb Expression Vectors to Obtain Stable CellLines

PER.C6 cells were seeded in DMEM plus 10% FBS in 47-tissue culturedishes (10 cm diameter) with approximately 2.5×10⁶ cells per dish andwere kept overnight under their normal culture conditions (10% CO₂concentration and 37° C.). The next day, co-transfections were performedin 39 dishes at 37° C. using Lipofectamine in standard protocols with 1μg MunI-digested and purified pUBS-Heavy2000/Hyg(−) and 1 μgSeal-digested and purified pUBS-Light2001/Neo (see, Example 3) per dish,while two dishes were co-transfected as controls with 1 μg MunI-digestedand purified pcDNA2000/Hyg(−) and 1 μg Seal-digested and purifiedpcDNA2001/Neo. As a control for transfection efficiency, four disheswere transfected with a LacZ control vector, while two dishes were nottransfected and served as negative controls.

After hours, cells were washed twice with DMEM and re-fed with freshmedium without selection. The next day, medium was replaced by freshmedium containing different selection reagents: 33 dishes of the heavyand light chain co-transfectants, two dishes that were transfected withthe empty vectors and the two negative controls (no transfection) wereincubated only with 50 μg per ml hygromycin, two dishes of the heavy andlight chain co-transfectants and two dishes of the transfectionefficiency dishes (LacZ vector) were incubated only with 500 μg per mlG418, while two transfection efficiency dishes were not treated withselection medium but used for transfection efficiency that was around40%. Two dishes were incubated with a combination of 50 μg per mlhygromycin and 250 μg per ml G418 and two dishes were incubated with 25μg per ml hygromycin and 500 μg per ml G418.

Since cells were overgrowing when they were only incubated withhygromycin alone, it was decided that a combination of hygromycin andG418 selection would immediately kill the cells that integrated only onetype of the two vectors that were transfected. Seven days after seeding,all co-transfectants were further incubated with a combination of 100 μgper ml hygromycin and 500 μg per ml G418. Cells were refreshed two orthree days with medium containing the same concentrations of selectingagents. Fourteen days after seeding, the concentrations were adjusted to250 μg per ml G418 and 50 μg per ml hygromycin. Twenty-two days afterseeding, a large number of colonies had grown to an extent in which itwas possible to select, pick and subculture. Approximately 300 separatecolonies were selected and picked from the 10 cm dishes and subsequentlygrown via 96 wells and/or 24 wells via six-well plates to T25 flasks. Inthis stage, cells are frozen (four vials per subcultured colony) andproduction levels of recombinant UBS-54 mAb are determined in thesupernatant using the ELISA described in Example 26.

CHO-dhfr cells are seeded in DMEM plus 10% FBS including hypoxanthineand thymidine in tissue culture dishes (10 cm diameter) withapproximately 1 million cells per dish and are kept overnight undernormal conditions and used for a co-transfection the next day withpUBS-Heavy2000/Hyg(−) and pUBS-Light2001/DHFRwt under standard protocolsusing Lipofectamine. Medium is replaced with fresh medium after a fewhours and split to different densities to allow the cells to adjust tothe selection medium when stable integration is taking place without apossible outgrowth of non-transfected cells. Colonies are first selectedon hygromycin resistance and, subsequently, MTX is added to select fordouble integrations of the 2 plasmids in these subcultured cell lines.

Transfections as described for pUBS-Heavy2000/Hyg(−) andpUBS-Light2001/Neo are performed with pUBS2-Heavy2000/Hyg(−) andpUBS2-Light2001/Neo in PER.C6 and PER.C6/E2A and selection is performedwith either subsequent incubation with hygromycin followed by G418 or asdescribed above with a combination of both selection reagents. CHO-dhfrcells are transfected with pUBS2-Heavy2000/Hyg(−) andpUBS2-Light2001/DHFRwt as described herein and selection is performed ina sequential way in which cells are first selected with hygromycin,after which an integration of the light chain vector is controlled byselection on MTX.

Furthermore, PER.C6 and PER.C6/E2A cells are also used for transfectionswith pUBS-3000/Hyg(−) and pUBS2-3000/Hyg(−), while CHO-dhfr cells aretransfected with pUBS-3000/DHFRwt and pUBS2-3000/DHFRwt, after which aselection and further amplification of the integrated plasmids areperformed by increasing the MTX concentration. In the case of thepcDNAs3000 plasmids, an equal number of mRNAs of both the heavy andlight chain is expected, while in the case of two separate vectors, itis unclear whether a correct equilibrium is achieved between the twosubunits of the immunoglobulin.

Transfections are also being performed on PER.C6, PER.C6/E2A andCHO-dhfr with expression vectors described in Examples 4 and 5 to obtainstable cell lines that express the humanized IgG1 mAb CAMPATH-1H and thehumanized IgG1 mAb 15C5 respectively.

Example 21 Sub-Culturing of Transfected Cells

From PER.C6 cells transfected with pUBS-Heavy2000/Hyg (−) andPUBS-Light2001/Neo, approximately 300 colonies that were growing inmedium containing Hygromycin and G418 were generally grown subsequentlyin 96-well, 24-well and 6-well plates in their respective medium plustheir respective selecting agents. Cells that were able to grow in24-well plates were checked for mAb production by using the ELISAdescribed in Example 26. If cells scored positively, at least one vialof each clone was frozen and stored, and cells were subsequently testedand subcultured. The selection of a good producer clone is based on highexpression, culturing behavior and viability. To allow checks for longterm viability, amplification of the integrated plasmids and suspensiongrowth during extended time periods, best producer clones are frozen, ofwhich a number of the best producers of each cell line are selected forfurther work. Similar experiments are being performed on CHO-dhfr cellstransfected with different plasmids and PER.C6 and PER.C6/E2A cells thatwere transfected with other combinations of heavy and light chains andother combinations of selection markers.

Example 22 mAb Production in Bioreactors

The best UBS-54 producing transfected cell line of PER.C6 is broughtinto suspension by washing the cells in PBS and then culturing the cellsin JRH ExCell 525 medium, first in small culture flasks and subsequentlyin roller bottles, and scaled up to 1 to 2 liter fermentors. Cells arekept on hygromycin and G418 selection until it is proven thatintegration of the vectors is stable over longer periods of time. Thisis done when cells are still in their attached phase or when cells arein suspension.

Suspension growing mAb producing PER.C6 cells are generally culturedwith hygromycin and G418 and used for inoculation of bioreactors fromroller bottles. Production yields, functionality and quality of theproduced mAb is checked after growth of the cells in perfusedbioreactors and in fed batch settings.

A. Perfusion in a 2 Liter Bioreactor.

Cells are seeded in suspension medium in the absence of selecting agentsat a concentration of approximately 0.5×10⁶ cells per ml and perfusionis started after a number of days when cell density reachesapproximately 2 to 3×10⁶ cells per ml. The perfusion rate is generally 1volume per 24 hours with a bleed of approximately 250 ml per 24 hours.In this setting, cells stay normally at a constant density ofapproximately 5×10⁶ cells per ml and a viability of almost 95% for overa month. The mAb production levels are determined on a regular basis.

B. Fed Batch in a 2 Liter Bioreactor.

In an initial run, mAb producing PER.C6 suspension cells that are grownon roller bottles are used to inoculate a 2 liter bioreactor in theabsence of selecting agents to a density of 0.3 to 0.5 million cells perml in a working volume of 300 to 500 ml and are left to grow until theviability of the cell culture drops to 10%. As a culture lifetimestandard, it is determined at what day after inoculation the viable celldensity drops beneath 0.5 million cells per ml. Cells normally growuntil a density of 2 to 3 million cells per ml, after which the mediumcomponents become limiting and the viability decreases. Furthermore, itis determined how much of the essential components, such as glucose andamino acids in the medium are being consumed by the cells. Next to that,it is determined what amino acids are being produced and what otherproducts are accumulating in the culture. Depending on this,concentrated feeding samples are being produced that are added atregular time points to increase the culture lifetime and therebyincrease the concentration of the mAb in the supernatant. In anothersetting, 10× concentrated medium samples are developed that are added tothe cells at different time points and that also increase the viabilityof the cells for a longer period of time, finally resulting in a higherconcentration of mAb in the supernatant.

Example 23 Transient Expression of Humanized Recombinant MonoclonalAntibodies

The correct combinations of the UBS-54 heavy and light chain genescontaining vectors were used in transient transfection experiments inPER.C6 cells. For this, it is not important which selection marker isintroduced in the plasmid backbone, because the expression lasts for ashort period (two to three days). The transfection method is generallylipofectamine, although other cationic lipid compounds for efficienttransfection can be used. Transient methods are extrapolated from T25flasks settings to at least 10-liter bioreactors. Approximately 3.5million PER.C6 and PER.C6/E2A cells were seeded at day 1 in a T25 flask.At day 2, cells were transfected with, at most, 8 μg plasmid DNA usinglipofectamine and refreshed after two to four hours and left for twodays. Then, the supernatant was harvested and antibody titers weremeasured in a quantitative ELISA for human IgG1 immunoglobulins (CLB,see also Example 26). Levels of total human antibody in this system areapproximately 4.8 μg/million seeded cells for PER.C6 and 11.1 μg/millionseeded cells for PER.C6/E2A. To determine how much of the producedantibody is of full size and built up from two heavy and two lightchains, as well as the expression levels of the heavy and/or light chainalone and connected by disulfide bridges, control ELISAs recognizing thesub-units separately are developed. Different capturing and stainingantibody combinations are used that all detect human(ized) IgG1sub-units. Supernatants of PER.C6 transfectants (transfected withcontrol vectors or pUBS-Heavy2000/Hyg(−) and pUBS-Light2001/DHFRwt) werechecked for full sized mAb production (FIG. 24) (of the incorporated'007 application). Samples were treated with and without DTT, whereinone can distinguish between full sized mAb (non-reduced) and heavy andlight chain separately (reduced). As expected, the heavy chain is onlysecreted when the light chain is co-expressed and most of the antibodyis of full size.

Example 24 Scale-Up System for Transient Transfections

PER.C6 and derivatives thereof are used for scaling up the DNAtransfections system. According to Wurm and Bernard (1999),transfections on suspension cells can be performed at 1 to 10 literset-ups in which yields of 1 to 10 mg/l (0.1 to 1 pg/cell/day) ofrecombinant protein have been obtained using electroporation.

A need exists for a system in which this can be well controlled andyields might be higher, especially for screening of large numbers ofproteins and toxic proteins that cannot be produced in a stable setting.Moreover, since cell lines such as CHO are heavily affected byapoptosis-inducing agents such as lipofectamine, the art teaches thatthere is a need for cells that are resistant to this. Since PER.C6 ishardly affected by transfection methods, it seems that PER.C6 andderivatives thereof are useful for these purposes. One to 50 litersuspension cultures of PER.C6 and PER.C6/E2A growing in adjusted mediumto support transient DNA transfections using purified plasmid DNA areused for electroporation or other methods, performing transfection withthe same expression plasmids. After several hours, the transfectionmedium is removed and replaced by fresh medium without serum. Therecombinant protein is allowed to accumulate in the supernatant forseveral days, after which the supernatant is harvested and all the cellsare removed. The supernatant is used for down stream processing topurify the recombinant protein.

Example 25 Scale Up System for Viral Infections

Heavy and light chain cDNAs of the antibodies described in Examples 3, 4and 5 are cloned into recombinant adenoviral adapter plasmids separatelyand in combination. The combinations are made to ensure an equalexpression level for both heavy and light chains of the antibody to beformed. When heavy and light chains are cloned separately, viruses arebeing produced and propagated separately, of which the infectability andthe concentration of virus particles are determined and finallyco-infected into PER.C6 and derivatives thereof to produce recombinantmAbs in the supernatant. Production of adapter vectors, recombinantadenoviruses and mAbs is as described for recombinant EPO (see, Examples13 and 14).

Example 26 Development of an ELISA for Determination of Human mAbs

Greiner microlon plates #655061 were coated with an anti-human IgG1kappa monoclonal antibody (Pharmingen #M032196 0.5) with 100 μl per wellin a concentration of 4 μg per ml in PBS. Incubation was performedovernight at 4° C. or for 90 minutes at 37° C. Then, wells were washedthree times with 0.05% Tween/PBS (400 μl per well) and subsequentlyblocked with 100 μl 5% milk dissolved in 0.05% Tween/PBS per well for 30minutes at 37° C. and then, the plate was washed three times with 400 μl0.05% Tween/PBS per well. As a standard, a purified human IgG1 antibodywas used (Sigma, #108H9265) diluted in 0.5% milk/0.05% Tween/PBS indilutions ranging from 50 to 400 ng per ml. Per well, 100 μl of thestandard was incubated for one hour at 37° C. Then, the plate was washedthree times with 400 μl per well 0.05% Tween/PBS. As the secondantibody, a biotin labeled mouse monoclonal anti-human IgG1 antibody wasused (Pharmingen #M045741) in a concentration of 2 ng per ml. Per well,100 μl of this antibody was added and incubated for one hour at 37° C.and the wells were washed three times with 400 μl 0.05% Tween/PBS.

Subsequently, conjugate was added: 100 μl per well of a 1:1000 dilutionof Streptavidin-HRP solution (Pharmingen #M045975) and incubated for onehour at 37° C., and the plate was again washed three times with 400 μlper well with 0.05% Tween/PBS.

One ABTS tablet (Boehringer Mannheim #600191-01) was dissolved in 50 mlABTS buffer (Boehringer Mannheim #60328501) and 100 μl of this solutionwas added to each well and incubated for one hour at RT or 37° C.Finally, the OD was measured at 405 nm. Supernatant samples from cellstransfected with mAb encoding vectors were generally dissolved anddiluted in 0.5% milk/0.05% Tween/PBS. If samples did not fit with thelinear range of the standard curve, other dilutions were used.

Example 27 Production of Influenza HA and NA Proteins in a Human Cellfor Recombinant Subunit Vaccines

cDNA sequences of genes encoding hemaglutinin (HA) and neuraminidase(NA) proteins of known and regularly appearing novel influenza virusstrains are being determined and generated by PCR with primers forconvenient cloning into pcDNA2000, pcDNA2001, pcDNA2002 and pcDNAs3000vectors (see, Example 1). Subsequently, these resulting expressionvectors are being transfected into PER.C6 and derivatives thereof forstable and transient expression of the recombinant proteins to result inthe production of recombinant HA and NA proteins that are, therefore,produced in a complete standardized way with human cells under strictand well-defined conditions. Cells are allowed to accumulate theserecombinant HA and NA proteins for a standard period of time. When thepcDNAs3000 vector is used, it is possible to clone both cDNAssimultaneously and have the cells produce both proteins at the sametime. From separate or combined cultures, the proteins are beingpurified following standard techniques and final HA and NA titers arebeing determined and activities of the proteins are checked by personsskilled in the art. Then, the purified recombinant proteins are used forvaccination studies and finally used for large-scale vaccinationpurposes.

The HA1 fragment of the swine influenza virus A/swine/Oedenrode/7C/96(Genbank accession number AF092053) was obtained by PCR using a forwardprimer with the following sequence: 5′ ATT GGC GCG CCA CCA TGA AGA CTATCA TTG CTT TGA GCT AC 3′ (SEQ ID NO:30) corresponding to theincorporated '007 application, and with a reverse primer with thefollowing sequence: 5′ GAT GCT AGC TCA TCT AGT TTG TTT TTC TGG TAT ATTCCG 3′ (SEQ ID NO:31) corresponding to the incorporated '007application. The resulting 1.0 kb PCR product was digested with AscI andNheI restriction enzymes and ligated with AscI and NheI-digested andpurified pcDNA2000/DHFRwt vector, resulting in pcDNA2001/DHFRwt-swHA1.Moreover, the HA2 fragment of the same virus was amplified by PCR usingthe same forward primer as described for HA1 and another reverse primerwith the following sequence: 5′ GAT GCT AGC TCA GTC TTT GTA TCC TGA CTTCAG TTC AAC ACC 3′ (SEQ ID NO:32) corresponding to the incorporated '007application. The resulting 1.6 kb HA2 PCR product was cloned in anidentical way as described for HA1, resulting in pcDNA2001/DHFRwt-swHA2.

Example 28 Integration of cDNAs Encoding Post-Translational ModifyingEnzymes

Since the levels of recombinant protein production in stable andtransiently transfected and infected PER.C6 and PER.C6/E2A are extremelyhigh and since a higher expression level is usually obtained upon DHFRdependent amplification due to increase of MTX concentration, an“out-titration” of the endogenous levels of enzymes that are involved inpost-translational modifications might occur.

Therefore, cDNAs encoding human enzymes involved in different kinds ofpost-translational modifications and processes such as glycosylation,phosphorylation, carboxylation, folding and trafficking are beingoverexpressed in PER.C6 and PER.C6/E2A to enable a more functionalrecombinant product to be produced to extreme levels in small and largesettings. It was shown that CHO cells can be engineered in which analpha-2,6-sialyltransferase was introduced to enhance the expression andbioactivity of tPA and human erythropoietin (Zhang et al., 1998, Minchet al., 1995, Jenkins et al., 1998). Other genes such as beta1,4-galactosyltransferase were also introduced into insect and CHO cellsto improve the N-linked oligosaccharide branch structures and to enhancethe concentration of sialic acids at the terminal residues (Weikert etal., 1999; Hollister et al., 1998). PER.C6 cells are modified byintegration of cDNAs encoding alpha 2,3-sialyltransferase, alpha2,6-sialyltransferase and beta 1,4-galactosyltransferase proteins tofurther increase the sialic acid content of recombinant proteinsproduced on this human cell line.

Example 29 Inhibition of Apoptosis by Overexpression of Adenovirus E1Bin CHO-dhfr Cells

It is known that CHO cells, overexpressing recombinant exogenousproteins, are highly sensitive for apoptotic signals, resulting in agenerally higher death rate among these stable producing cell lines ascompared to the wild-type or original cells from which these cells werederived. Moreover, CHO cells die of apoptotic effects when agents suchas lipofectamine are being used in transfection studies. Thus, CHO cellshave a great disadvantage in recombinant protein production in the sensethat the cells are very easily killed by apoptosis due to differentreasons. Since it is known that the E1B gene of adenovirus hasanti-apoptotic effects (White et al., 1992; Yew and Berk 1992), stableCHO-dhfr cells that express both heavy and light chains of the describedantibodies (see, Examples 3, 4 and 5) are being transfected withadenovirus E1B cDNAs to produce a stable or transient expression of theE1B proteins to finally ensure a lower apoptotic effect in these cellsand thereby increase the production rate of the recombinant proteins.Transiently transfected cells and stably transfected cells are comparedto wild-type CHO-dhfr cells in FACS analyses for cell death due to thetransfection method or due to the fact that they over-express therecombinant proteins.

Stable CHO cell lines are generated in which the adenovirus E1B proteinsare overexpressed. Subsequently, the apoptotic response due to effectsof, for instance, Lipofectamine in these stable E1B producing CHO cellsis compared to the apoptotic response of the parental cells that did notreceive the E1B gene. These experiments are executed using FACS analysesand commercially available kits that can determine the rate ofapoptosis.

Example 30 Inhibition of Apoptosis by Overexpression of Adenovirus E1Bin Human Cells

PER.C6 cells and derivatives thereof do express the E1A and E1B genes ofadenovirus. Other human cells, such as A549 cells, are being used tostably overexpress adenovirus E1B to determine the anti-apoptoticeffects of the presence of the adenovirus E1B gene as described for CHOcells (see, Example 29). Most cells do respond to transfection agentssuch as lipofectamine or other cationic lipids, resulting in massiveapoptosis and finally resulting in low concentrations of the recombinantproteins that are secreted, simply due to the fact that only few cellssurvive the treatment. Stable E1B overexpressing cells are compared tothe parental cell lines in their response to overexpression of toxicproteins or apoptosis inducing proteins and their response totransfection agents such as lipofectamine.

Example 31 Generation of PER.C6 Derived Cell Lines Lacking a FunctionalDHFR Protein

PER.C6 cells are used to knock out the DHFR gene using different systemsto obtain cell lines that can be used for amplification of the exogenousintegrated DHFR gene that is encoded on the vectors that are describedin Examples 1 to 5 or other DHFR expressing vectors. PER.C6 cells arescreened for the presence of the different chromosomes and are selectedfor a low copy number of the chromosome that carries the human DHFRgene. Subsequently, these cells are used in knock-out experiments inwhich the open reading frame of the DHFR gene is disrupted and replacedby a selection marker. To obtain a double knock-out cell line, bothalleles are removed via homologous recombination using two differentselection markers or by other systems as, for instance, described forCHO cells (Urlaub et al., 1983).

Other systems are also applied in which the functionality of the DHFRprotein is lowered or completely removed, for instance, by the use ofanti-sense RNA or via RNA/DNA hybrids, in which the gene is not removedor knocked out, but the down stream products of the gene are disturbedin their function.

Example 32 Long-Term Production of Recombinant Proteins Using Proteaseand Neuraminidase Inhibitors

Stable clones described in Example 8 are used for long-term expressionin the presence and absence of MTX, serum and protease inhibitors. Whenstable or transfected cells are left during a number of days toaccumulate recombinant human EPO protein, a flattening curve instead ofa straight increase is observed, which indicates that the accumulatedEPO is degraded in time. This might be an inactive process due toexternal factors such as light or temperature. It might also be thatspecific proteases that are produced by the viable cells or that arereleased upon lysis of dead cells digest the recombinant EPO protein.Therefore, an increasing concentration of CuSO₄ is added to the culturemedium after transfection and on the stable producing cells to detect amore stable production curve. Cells are cultured for several days andthe amount of EPO is determined at different time points. CuSO₄ is aknown inhibitor of protease activity, which can be easily removed duringdown stream processing and EPO purification. The most optimalconcentration of CuSO₄ is used to produce recombinant human EPO proteinafter transient expression upon DNA transfection and viral infections.Furthermore, the optimal concentration of CuSO₄ is also used in theproduction of EPO on the stable clones. In the case of EPO in which thepresence of terminal sialic acids is important to ensure a longcirculation half-life of the recombinant protein, it is necessary toproduce highly sialylated EPO. Since living cells produce neuraminidasesthat can be secreted upon activation by stress factors, it is likelythat produced EPO lose their sialic acids due to these stress factorsand produced neuraminidases. To prevent clipping off of sialic acids,neuraminidase inhibitors are added to the medium to result in aprolonged attachment of sialic acids to the EPO that is produced.

Example 33 Stable Expression of Recombinant Proteins in Human CellsUsing the Amplifiable Glutamine Synthetase System

PER.C6 and derivatives thereof are being used to stably expressrecombinant proteins using the glutamine synthetase (GS) system. First,cells are being checked for their ability to grow in glutamine-freemedium. If cells cannot grow in glutamine-free medium, this means thatthese cells do not express enough GS, finally resulting in death of thecells. The GS gene can be integrated into expression vectors as aselection marker (as is described for the DHFR gene) and can beamplified by increasing the methionine sulphoximine (MSX) concentrationresulting in overexpression of the recombinant protein of interest,since the entire stably integrated vector will be co-amplified as wasshown for DHFR. The GS gene expression system became feasible after areport of Sanders et al. (1984) and a comparison was made between theDHFR selection system and GS by Cockett et al. (1990). The production ofrecombinant mAbs using GS was first described by Bebbington et al.(1992).

The GS gene is cloned into the vector backbones described in Example 1or cDNAs encoding recombinant proteins and heavy and light chains ofmabs are cloned into the available vectors carrying the GS gene.Subsequently, these vectors are transfected into PER.C6 and selectedunder MSX concentrations that will allow growth of cells with stableintegration of the vectors.

Example 34 Production of Recombinant HIV gp120 Protein in a Human Cell

The cDNA encoding the highly glycosylated envelope protein gp120 fromHuman Immunodeficiency Virus (HIV) is determined and obtained by PCRusing primers that harbor a perfect Kozak sequence in the upstreamprimer for proper translation initiation and convenient restrictionrecognition sequences for cloning into the expression vectors describedin Example 1. Subsequently, this PCR product is sequenced on bothstrands to ensure that no PCR mistakes are being introduced.

The expression vector is transfected into PER.C6, derivatives thereofand CHO-dhfr cells to obtain stable producing cell lines. Differences inglycosylation between CHO-produced and PER.C6 produced gp120 are beingdetermined in 2D electrophoresis experiments and subsequently in MassSpectrometry experiments, since gp120 is a heavily glycosylated proteinwith mainly O-linked oligosaccharides. The recombinant protein ispurified by persons skilled in the art and subsequently used forfunctionality and other assays. Purified protein is used for vaccinationpurposes to prevent HIV infections.

Example 35 Transient Expression of Recombinant Proteins on PER.C6 CellsMediated by E1A/E2A-Deleted Ad5 Infection

Transient production of recombinant proteins using plasmid DNA in celllines in suspension is currently performed using for example CaPO₄co-precipitation or PEI (poly-ethylenimine) reagents. The transfectionefficiency and consequently protein yield are in general low and rangebetween a few to 25 mg/l at ≧1 liter scale. In the examples below atransient expression system is presented that makes use of recombinantadenoviruses that are replication deficient in the cell line used forthe production of proteins. The transient expression system is veryefficient allowing for easy up-scaling to 10 liters and further whenused in suspension cells. In these examples, the recombinant Ad5-basedadenoviruses are deleted for the E1 and E2A regions and for that reasoncan only be generated on cell lines that provide both functions, as forexample PER.E2A cells (see U.S. Pat. No. 6,395,519).

Recombinant viruses that are both E1 and E2A deleted were generated by ahomologous recombination procedure using a plasmid-based system asdescribed in U.S. Pat. No. 6,878,549. This system consists of a firstconstruct called adapter plasmid (pAdApt) that contains Ad5 sequences 1to 454 including the left ITR and packaging signal, an expressioncassette replacing the E1 gene, and an Ad5 fragment corresponding tonucleotides 3511 to 6095. The expression cassette consists of the humanCMV promoter, a multiple cloning site (MCS) and the SV40 pA. Nucleicacid encoding the protein of interest was cloned in the MCS.Furthermore, the plasmid-based system consisted of a cosmid(pWE/Ad.AflII-rITRdE2A, see example 10 of U.S. Pat. No. 6,878,549)constituting Ad5 sequences between nucleotide 3534 and 35935 having adeletion encompassing the E2A coding sequences (corresponding tonucleotides 22443-24033; sequence numbering as in Ad5 genbank sequenceM73260) and that may be deleted for E3 sequences between Ad5 nucleotide28592 to 30471.

To evaluate Ad5.dE1.dE2A-mediated protein expression on PER.C6 cells weselected the following transgenes, representing three classes ofproteins of interest:

-   1. ACE2, the cellular receptor for SARS coronavirus (SARS-CoV) and    S1-spike, the viral spike protein of SARS-CoV both representing    membrane proteins,-   2. s.ACE2 and S565, the soluble forms of ACE2 and S1-spike    respectively belonging to the class of secreted proteins,-   3. SARS-AB; an antibody against SARS-CoV spike protein (ter Meulen    et al, Lancet, 2004; 363(9427): 2139-41).    Generation of Various Adapter Plasmids

In order to produce E1.E2A-deleted recombinant adenoviruses using thisplasmid system, first several pAdApt plasmids comprising the transgenes,as listed above, were constructed using conventional molecularbiological techniques.

Generation of pAdApt.S1-Spike and pAdApt.S565

The cDNA of S1-spike (3767 bp; J. Virol. 2005; 79(3):1635-1644.) wascodon-optimised for optimal expression in human cells by GENEART(Regensburg, Germany) and cloned into the expression cassette of pAdApt,resulting in pAdApt.S1-spike.

pAdApt.S565, the adapter plasmid containing the truncated form lackingthe trans-membrane region (referred to as ‘secreted’ or ‘soluble’ form)of S1-spike was generated after subcloning into the pAdApt-expressioncassette of a restriction fragment comprising the N terminal 1695 bp ofS1-spike. S565, a fragment containing the region responsible for bindingto ACE2 was fused in frame to a myc/his tag previously introduced inpAdApt, enabling purification and detection of the expressed protein.

Generation of pAdApt.ACE2 and pAdApt.sACE2

The cDNA encoding the full-length cellular receptor for SARS-CoV (ACE2;2417 bp; Genbank sequence AF291820) was synthesized by GENEART andcloned into the expression cassette of pAdApt, resulting in pAdApt.ACE2.

s.ACE2, the truncated variant of ACE2 lacking the transmembrane region(aa residues 741-805 of ACE2) was generated by cloning the 2217 bpcoding region (corresponding to residues 1 to 739 of ACE2) into theexpression cassette of pAdApt in frame with and upstream of a myc/histaq. The final plasmid was named pAdApt.sACE2.

Generation of pAdApt.SARS-AB

Genes encoding the variable heavy (VH) and variable light chain (VL) ofscFv 03-014; an antibody against the viral spike protein (S1) ofSARS-CoV (ter Meulen et al, Lancet, 2004; 363(9427): 2139-41)) wereconstructed synthetically by GENEART. These genes were separatelysubcloned into plasmids that already contained an appropriate leadersequence and either the constant CH1, CH2 and CH3 regions of the heavychain (HC) or Ckappa, the constant region of the light chain (LC)resulting in subclones containing the complete (constant and variable)SARS-AB HC and SARS-AB LC respectively. To enable translation of HC andLC from one transcript, a fragment including an Internal Ribosome EntrySite (IRES) sequence was inserted in between HC and LC and cloned in theexpression cassette of pAdApt (thereby replacing the original CMVpromoter for a CMV promoter containing an additional chimeric intron).Initially identical leader sequences were used upstream the heavy andlight chains. In order to circumvent vector instability due tohomologous recombination between these identical leaders during virusreplication, the leader of the LC was exchanged for another one (sharing44% homology on nucleotide level) that was demonstrated to be equallyfunctional. The final construct was named pAdApt.SARS-AB.

Virus Generation, Production and Purification.

Generation of recombinant Adenovirus batches with E1 and E2A deleted(Ad5.dE1.dE2A) was accomplished using a lipofectamine mediatedco-transfection procedure using 2 μg linearized (Pac I/Sal I digested)pAdApt carrying the various transgenes and 6 μg linearized (Pac Idigested) cosmid (pWE/AflII-rITRdE2A). Co-transfection of adherentPER.E2A cells was performed using Lipofectamine-2000 according tomanufacturers instructions. Following co-transfection cells werecultured in 25 cm² flasks and after two days the culture format wasexpanded to 80 cm² scale. Upon signs of full cyto-pathic effect (CPE)indicating that functional adenovirus has formed, cells and medium wereharvested and recombinant virus was released by freeze-thawing,centrifuged to remove cell debris and supernatants were collected. Thesecleared supernatants are referred to as crude lysates.

To generate high quality purified viruses, crude lysates weresubsequently used to plaque purify the recombinant viruses according tostandard methods. A number of isolated plaques for each virus wereisolated and amplified on PER.E2A cells in 24 well plates to generatecrude lysates for analysis. All plaques were tested for virus integrityand identity by PCR analysis of the transgene, E2A and the E3 region.Based on these results PER.E2A host cells were infected with part of theplaque-amplified material and grown at 34° C. until full CPE wasobserved. Cells and medium were harvested and stored at −20° C. Crudelysates were prepared and part of this material was used for infectionin subsequent steps. In this manner the virus was expanded fromamplified plaque (1 ml) to 25 cm² flask (5 ml), 175 cm² flask (15 ml)and finally to a triple layer flask (175III cm², 50 ml). In a productionrun (48×175III cm²) PER.E2A cells were infected using the supernatant ofthe triple layer as inoculum. Productions were harvested when CPE wasvisible and virus was purified from the packaging cells using a two-stepCsCl banding procedure. Purification of intact virus particles fromremains (protein and fragmented DNA) of the host cell was performedusing a second CsCl gradient centrifugation.

Purified virus batches were tested for the presence of intact transgene,and presence or absence of E3 and E2A regions respectively. Transgeneexpression and both titer and infectious titer were determined usingstandard western blot, High Pressure Liquid Chromatography (HPLC) andTissue Culture Infectious Dose 50 (TCID₅₀) assays respectively. In allvirus batches intact transgenes, presence of E3 and absence of E2Aregions were confirmed by PCR using specific primers.

Transgene expression of all batches was tested in adherent PER.C6cultures after two days of infections using MOI's ranging from 0 to 3000vp/cell. Presence in the culture medium of (myc/his-tagged) S565 ands.ACE2 was demonstrated by western blot using mouse-anti-c.myc(HRP-conjugated), while SARS-AB was detected on western blot usingdonkey-anti-human IgG (HRP-conjugated). Proper expression of thetransmembrane proteins S1-spike and ACE2 on the cell membranes weredetected using FACS after cell staining with human-anti-spike(CR3014)/goat-anti-human IgG (PE-conjugated) for S1-spike andgoat-anti-ACE2/donkey anti IgG (PE-conjugated) for ACE2.

Instead of generating purified batches of Ad5.dE1.dE2A viruses, thecrude lysates can be directly used to inoculate PER.C6 cells and producerecombinant proteins. The crude lysate can also be first amplified onPER.E2A cells grown in larger formats to generate more crude lysate,enabling inoculation of a larger volume with more PER.C6 cells forprotein production.

Transient Expression of Proteins in PER.C6 Cells

To study protein synthesis in PER.C6 cells, virus batches were producedon PER-E2A cells as described above, and used to infect PER.C6 cells inroller bottles and shaker flasks according to the method outlined below.For infection of PER.C6 cells in roller bottles (100 ml culture volume)cells were seeded at 0.3×10⁶ cells/ml in AEM medium (Invitrogen)supplemented with L-glutamine. Cells were cultured in roller bottles (2rpm) at 37° C. in an incubator at 10% CO₂. When cell concentrationsreached 2×10⁶ cells/ml, cultures were centrifuged for 10 minutes at500×g and pellets were resuspended in 100 ml fresh AEM/L-glutamine at afinal cell concentration of 2.5×10⁶ cells/ml. The next day cells werecounted using the Casey counter and virus was added to the rollerbottles corresponding to the desired MOI.

For infection of PER.C6 cells in shaker flasks (25 ml culture volume)cells grown in roller bottles (as above) were counted, centrifuged andcell pellets were resuspended in fresh AEM/L-glutamine at 2.5×10⁶cells/ml after which virus was added at the desired MOI. Cells werecultured in shaker flasks (75 rpm) at 37° C. at 10% CO₂.

Harvest of the medium was done between day 3 and 14, depending on thetype of experiment and the protein expressed. The method described abovefor roller bottles and shaker flasks can simply be adapted and appliedfor other culture formats by persons skilled in the art. Also thementioned cell densities and medium can be modified to achieve higherproduction levels.

Example 36 Yields of Various Recombinant Proteins Produced in PER.C6Cells Mediated by Ad5.dE1.dE2A Infection

The following experiments were performed to define preferred parameterslike multiplicity of infection (MOI), cell density and day of harvestfor Ad5.dE1.dE2A-mediated protein synthesis in PER.C6 cells. Inaddition, typical yields of the secreted proteins (s.ACE2 and S565) andthe yield of SARS-AB obtained in PER.C6 cells mediated by E1.E2A-deletedAd5 infection is presented.

In order to determine the preferred MOI for protein yield, PER.C6 cellswere infected with Ad5.dE1.dE2A.S565 at various MOI's, medium containingS565 protein was collected and quantified by western blot.

Hereto, cells (1×10⁶ cells/ml) were cultured and infected in shakerflasks (25 ml) in AEM medium according to the procedure described above(example 35). Cells were infected with Ad5.dE1.dE2A.S565 at MOI's of 0,100, 250, 500, 1000, 1500 and 2000 vp/cell, and cultured for 5 days inAEM. Every day samples were taken from these cultures, centrifuged andthe pellets were resuspended in 1 ml PBS. In these samples numbers oftotal cells and viable cells were measured using the easy counteraccording to a standard procedure. The supernatants were filtered,proteins were separated by SDS-PAGE and analyzed in western blots usinganti-myc monoclonal antibody (FIG. 2). Western blot analysis of samples,taken from day 2 and day 3 after infection with MOI 100, showed onesingle product of the expected molecular weight of 97 kD. Samples takenfrom cultures that were infected with the higher (>100) MOI's showed,besides the product of the expected size, an additional product around64 kD of unknown origin. Based on the presence of this extra fragment,MOI's 250, 500, 1000, 1500 and 2000 vp/cell were considered not to beoptimal for further experimental use. Moreover, the amount of producedprotein did not increase with the increasing MOI, indicating that therewas an excess of virus. These observations indicated that the MOI ispreferably <100 vp/cell, and a new infection experiment using MOI's ofless then 100 vp/ml was initiated.

Lower MOI's as well as multiple cell densities were included in the nextexperiment, in order to determine both preferred MOI as well aspreferred cell concentrations for protein production in PER.C6 cells.Various PER.C6 cell cultures (1×10⁶, 2×10⁶ and 2.5×10⁶ cells/ml) wereinfected with MOI's of 0, 30, 60 and 100 vp/cell. Samples were collectedand analyzed similarly as described above. Samples taken on days 2, 3and 4 were analyzed in western blots and showed the highest signal forMOI 30 at a cell density of 2.5×10⁶ cells/ml although differencesbetween infection with MOI 30 and 60 were within a factor of 2-3.

Similar infection experiments in roller bottles in BMIV medium (aproprietary medium of Crucell Holland B.V.) using MOI of 30 and a celldensity of 2.5×10⁶ cells/ml during 7 days confirmed highest productyields on day 4-post infection.

To investigate if MOI 30 also gave good results for expressing anantibody, a similar experiment was performed whereby PER.C6 cells(2.5×10⁶ cells/ml) were infected with Ad5.dE1.dE2A.SARS-AB at MOIs of 0,30, 60, 120 and 240 vp/cell. Cultures were monitored and sampled for 8days. SARS-AB yield was quantified using a standardized AKTA-ProtAmethod. Increasing SARS-AB concentrations were detected in all culturesover time with final values of 23, 18, 10 and 6 μg/ml on day 8 incultures infected with MOI 30, 60, 120 and 240 vp/cell respectivelyshowing highest antibody production levels following infection with 30vp/cell (FIG. 3).

These experiments thus demonstrated efficient protein production inPER.C6 cells in suspension cultures using the E1-E2A-deletedadenoviruses, and led to the surprising observation that best yieldswere obtained with the lower range of MOIs.

Product Yield and Purity Following E1/E2a-Deleted Adenovirus-MediatedTransient Expression in PER. C6 Cells

To determine the yields of the three types of secreted proteins, S565,s.ACE2 and SARS-AB, experiments were performed using the conditionsdetermined above.

PER.C6 roller bottle cultures (100 ml) were infected with MOI 30 and 60vp/cell at 2.5×10⁶ cells/ml as described above. In case of S565 ands.ACE2, cells were cultured in parallel for 4 days post infection, bothin AEM and in BMIV medium. Ad5.dE1.dE2A.SARS-AB infected cells werecultured for 12 days in AEM. Harvested culture media were centrifugedand filtered over a 0.22 μm bottle top filter.

In case of S565 and s.ACE2, total protein concentration in cultureharvests was determined using the Micro BCA™ protein assay (Pierce; TheNetherlands), according to manufacturers protocol. Samples of theharvests were also serially diluted and separated on SDS-PAGE gels.After colloid blue staining, the gel was optically scanned and allsingle products (in the linear sample range) present in the individuallanes were digitally integrated and converted to Integrated OpticalDensity (IOD) values. This enabled the expression of the individualproteins as percentage of total protein per lane and is defined for thisexample as protein purity. Subsequently, the amount of produced S565 ands.ACE2 was calculated by multiplication of the purity by the totalamount of protein. In this way purities of the Ad5-mediated S565 ands.ACE2 in culture harvests were determined, and ranged between 60-75%and 30-40% respectively. These figures demonstrate that the describedmethod herein for transient protein expression is an efficient and cleansystem to produce proteins. Protein yields were calculated and rangedbetween 500-600 μg/ml for s.ACE2 and 220-370 μg/ml for S565. Yields arepresented in Table 2.

The SARS-AB concentration present in culture medium was determined usingHPLC-protA column chromatography standardized for IgG quantitationaccording to manufacturers protocol, and ranged between 120-140 μg/ml.

Analysis of Purified S565, s.ACE2 and SARS-AB

The secreted proteins S565 and s.ACE2 were chromatographically purifiedusing standard affinity and desalting columns. The soluble (Myc/Histagged) proteins S565 and s.ACE2 were produced in 100 ml roller bottles;expressed proteins present in culture medium were purified on an AKTAexplorer (GE-health care) using HisTrap HP (1 ml) affinity and HiTrapdesalting (5 ml) columns (GE-health care). Purified proteins werequantified by a micro BCA protein assay (Pierce; The Netherlands),according to manufacturers protocol.

SARS-AB was purified using HiTrap rProtein A protA and HiPrep 26/10affinity and desalting columns (GE-health care) and quantified byoptical density measurements at 280 nm.

Western blot analysis following denaturing SDS-PAGE of sets of seriallydiluted samples using a mouse-anti-c-myc (HRP-conjugated) MAb fordetection of s.ACE2 and S565, and a donkey-anti-human IgG(HRP-conjugated) MAb for detection of SARS-AB, demonstrated the presenceof only the products of the expected sizes (FIG. 4), indicatingefficient full length product formation and the absence of truncation ordegradation of products during synthesis or storage.

SARS-AB Production in PER. C6 Cells Using Crude Lysates

Transient expression of recombinant protein is aimed at the fastproduction of recombinant proteins in quantities enabling furthertesting in vitro and in vivo. The experiments described above wereperformed using purified and quantified adenovirus batches, which takeabout 3-4 months to complete.

In order to reduce the period of time required for Ad5-mediatedtransient expression it is desirable to use viruses from crude stocksearly in the procedure of virus generation. The next experimentdescribes the comparison of SARS-AB yield in PER.C6 cells afterinfection with either Ad5.dE1.dE2A.SARS-AB purified virus or crudelysate (harvested medium containing virus at the moment full-CPEoccurred following co-transfection of PER.E2A with pAdApt.SARS-AB andpWE/AflII-rITRdE2A). The two inoculums were referred to in this exampleas ‘purified virus’ and ‘crude lysate’ respectively. The crude lysate isalso referred to in the invention as a ‘crude adenovirus particlecompostion’.

Since the virus titer of the crude lysate cannot be accurately measuredusing standard HPLC methods, infectious titers (IU/ml) were determinedusing TCID₅₀ assays. Generating the crude lysate virus preparationincluding the TCID₅₀ determination takes around 4 weeks, representing aconsiderable time reduction as compared to the 3-4 months necessary togenerate the purified virus batch. In a side-by-side comparison study,PER.C6 cells were infected with purified virus or crude lysatepreparations using similar amounts of infectious virus particles (IU),corresponding to an MOI of 30 vp/ml of the purified batch. Cell cultureand virus infections of 100 ml roller bottles were performed asdescribed above. The number of total and viable cells was monitored for12 days post infection. Medium samples were collected and measured forSARS-AB production as described above. As shown in FIG. 5, bothinfections showed increasing SARS-AB concentration during the samplingperiod with higher product yields for cultures infected with crudelysate virus preparation (120 μg/ml) compared to the ones infected withthe purified virus (80 μg/ml). SARS-AB generated by both types of virusinoculums was harvested on day 12 and purified using AKTA-protAchromatography. Quality of the SARS-AB batches was analyzed usingnon-reduced colloid blue stained SDS-PAGE (FIG. 6), according tostandard protocols.

Both batches generated from purified adenovirus or from crude lysatepreparations showed identical patterns compared with the positivecontrol (the SARS-antibody from a stably transfected PER.C6 cell line(CR3014)).

This experiment thus demonstrates that quality and quantity of theSARS-AB produced using similar low infectious doses (MOI 30=4 IU/cell)of crude lysates and purified virus were comparable with productsisolated from a stable expressing PER.C6-derived cell line. Thus it ispossible to use crude lysates in the transient expression methoddescribed herein, which significantly reduces the time lines fortransient production of proteins.

Example 37 Functional Analysis of Virus Mediated Transiently ProducedProteins

In the next experiments transiently produced proteins produced accordingto the methods described in example 36 were analyzed for having properstructural conformation by testing specific interaction with a relevantligand or target in binding assays.

Proper expression and structural integrity of the membrane boundproteins ACE2, S1-spike and the secreted protein s.ACE2 was measuredusing FACS as outlined below.

Correct protein structure of transiently produced S565 and SARS-AB weretested in an ELISA-based assay for their ability to bind to CR3014 andto S1-spike, respectively. CR3014 is used as a positive control antibodyand is isolated from a PER.C6-derived cell line expressing the SARS-ABin a stable manner.

Functional Analysis of S565

Functionality of S565 was analyzed in an ELISA-based assay. Seriallydiluted purified myc/his-tagged S565 was bound to mouse-anti-c.myccoated 96-wells plates, and subsequently incubated with a fixed CR3014(anti-SARS Ab) concentration. Binding of CR3014 to S565 was detectedusing peroxidase-conjugated AffiniPure donkey anti-human IgG (JacksonImmuno Research), according to standard methods. The relationshipbetween optical density measured at 492 nm and the S565 concentrationshowed an S-curve representative for specific interaction between anantibody and its substrate. The result of this assay demonstrated theability of control antibody CR3014 to bind to the transiently producedand purified S565, indicating proper folding of this secreted protein(FIG. 7A).

Functional Analysis of SARS-AB

The purified S565 protein (of which the integrity was confirmed, seeprevious paragraph) preparation was used to analyze the two purifiedSARS-AB batches produced in PER.C6 cells, infected with purified virusor crude lysates of Ad5.dE1.dE2A.SARS-AB (described in example 36). Tothis end a fixed amount of purified myc/his-tagged S565 was linked tomouse-anti-c.myc-coated 96-wells plates, and subsequently incubated withtransiently produced SARS-AB in serial dilutions. Binding of SARS-AB toS565 was detected using peroxidase-conjugated AffiniPure donkeyanti-human IgG (Jackson Immuno Research) according to standard methods.S-curves generated in this binding assay demonstrated the ability of thetested SARS-AB batches to bind the immobilized S565 (FIG. 7B). Thus, theantibody produced using the method of the invention was functional inbinding its target.

Functional Analysis of S1-Spike

Transiently produced SARS-AB (functionality verified, see previousparagraph), was used to confirm proper conformation of S1-spike, thefull length SARS spike protein, in a FACS assay. Hereto, suspensionPER.C6 cells were infected with MOI 30 and MOI 100 vp/cell ofAd5.dE1.dE2A.S1-spike. Two days post-infection, cells were harvested andincubated with SARS-AB. The amount of SARS-AB bound to transientlyexpressed S1-spike on PER.C6 cell membranes was detected by incubationwith goat-anti-human-IgG (PE-conjugated) and analyzed by FACS. Detectionof positive cell populations of 95% and 91% by FACS analysis for MOI 30and 100 vp/ml respectively, demonstrated binding of SARS-AB to S1-spike.Thus, S1-spike had a proper conformation when transiently produced inthe Ad5.dE1.dE2A/PER.C6 protein production platform (FIG. 8).

Functional Analysis of s.ACE2

Transiently expressed S1.spike (proper conformation demonstrated, seeprevious paragraph) was used in a functional assay for s.ACE2. Properconformation of s.ACE2 was determined by its ability to interact withS1-spike and measured by FACS. Briefly, suspension PER.C6 cells wereinfected with 30 vp/cell of Ad5.dE1.dE2A.S1-spike and cultured for twodays, then cells were harvested and s.ACE2 (40 μg/ml) was added to thecells. The amount of s.ACE2 bound to membranes containing transientlyexpressed S1-spike was detected by incubating the cells withmouse-anti-c-Myc-FITC and analyzed using FACS. Detection of a positivecell population of 70% by FACS demonstrated the ability of s.ACE2 tobind to S1-spike and therefore demonstrated proper conformation ofvirus-mediated transiently produced s.ACE2 in PER.C6 cells (FIG. 9).

Functional Analysis of ACE2

Expression and structural integrity of the transmembrane ACE2 proteinwere analyzed by FACS, using a commercially available monoclonalantibody. Hereto, shaker flasks (25 ml; 2.5×10⁶ cells/ml) and rollerbottles (100 ml; 2.5×10⁶ cells/ml) were infected in parallel withAd5.dE1.dE2A.ACE2 at 0, 30, 60 and 120 vp/cell and harvested two daysfollowing infection. Cells were incubated subsequently withgoat-anti-human ACE-2 IgG and PE-conjugated anti-human IgG (100-folddilution) and analyzed using FACS-Calibur. In both culture formats,80-96% of positive cells were measured for various MOI's and confirmedproper expression of ACE2 on the cell surface of PER.C6 cells followingtransient expression. In addition, it also demonstrated that shakerflasks and roller bottles were infected with similar high efficiency(FIG. 10).

Tables

TABLE 1 moi ratio (virus (virus EPO yields particles particles/ (ELISAper infectious culture culture units/1E6 cell) units) conditions mediumrefreshment cells/day) 200 330 roller JRH day 3 240 bottle 200 330roller JRH none 190 bottle 20 330 roller JRH day 3 80 bottle 20 330roller JRH none 70 bottle 200 560 6-wells DMEM + every day 60 FBS

(=Table 5 of the incorporated '409 application).

EPO yields obtained after viral infections. Yields per million seededcells were determined with an EPO ELISA on supernatants from PER.C6cells that were infected with recombinant IG.Ad5.AdApt.EPO.dE2Aadenovirus as described in Example 14. Two different batches of thevirus were used with different vp/IU ratios (330 and 560) in twodifferent settings (roller bottle suspension cultures and 6-wellsattached cultures).

TABLE 2 Volumetric Specific production Protein Medium MOI (μg/ml)(pg/cell/day) s.ACE2 AEM 60 501 50

Volumetric and specific protein production levels in PER.C6 cellsfollowing virus-mediated production of various proteins. Cells werecultured in AEM or BMIV medium in roller bottles (100 ml; 2.5×10⁶cells/ml) and infected with MOI's of 30 or 60 vp/cell Ad5.dE1.dE2Avectors expressing s.ACE2, S565 or SARS-AB. See example 36 for details.

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1. A method for producing a protein of interest, the method comprising:a) providing a recombinant adenoviral vector comprising nucleic acidencoding the protein of interest under control of a promoter, whereinsaid recombinant adenoviral vector has deletions in the E1 and the E2Aregions of the adenovirus genome, b) propagating said recombinantadenoviral vector in complementing cells that express adenoviral E1protein and adenoviral E2A protein, to obtain recombinant adenovirusparticles, c) infecting a culture of complementing cells that expressadenoviral E1 protein, but not adenoviral E2A protein, with saidrecombinant adenovirus particles, to produce the protein of interest,and d) harvesting the protein of interest.
 2. The method according toclaim 1, wherein the complementing cells in step c) comprise PER.C6cells as deposited at the ECACC under number
 96022940. 3. The methodaccording to claim 1, wherein the complementing cells in step b)comprise PER.C6 cells that express the temperature sensitive 125 mutantof E2A (ts125 E2A).
 4. The method according to claim 1, wherein thepromoter is a cytomegalovirus (CMV) promoter.
 5. The method according toclaim 1, wherein steps b) and c) are performed in different culturevessels.
 6. The method according to claim 1, wherein in step c) theinfection of the complementing cells with the recombinant adenovirusparticles is performed at a multiplicity of infection (moi) of between 1and 1000 virus particles (vp)/cell.
 7. The method according to claim 6,wherein said moi is between 10 and 100 vp/cell.
 8. The method accordingto claim 1, wherein the recombinant adenovirus particles in step b) areobtained in a culture medium, and wherein the culture medium containingthe recombinant adenovirus particles is separated from the complementingcells to obtain a crude adenovirus particle composition, and whereinsaid crude adenovirus particle composition is used for the infection ofstep c).
 9. The method according to claim 1, wherein the recombinantadenovirus particles in step b) are purified, and subsequently used forthe infection of step c).
 10. The method according to claim 1, whereinthe protein of interest is an antibody.
 11. The method according toclaim 1, wherein the protein of interest is a blood coagulation protein.12. The method according to claim 1, wherein the protein of interest isa viral protein other than an adenovirus protein.
 13. The methodaccording to claim 12, wherein the protein of interest is influenzahemaglutinin (HA).
 14. The method according to claim 1, wherein theprotein of interest is erythropoietin (EPO).
 15. The method according toclaim 1, wherein the complementing cells in step c) are in suspension.16. A method of producing a protein of interest, the method comprising:providing a recombinant adenoviral vector comprising nucleic acidencoding the protein of interest under control of a promoter, whereinsaid recombinant adenoviral vector has deletions in a first region andin a second region of the adenovirus genome, wherein each of said firstsecond regions is required for adenoviral genome replication and/oradenovirus particle formation; propagating said recombinant adenoviralvector in a first type of complementing cells that expresses proteinsfrom said first and from said second regions of the adenovirus genome soas to complement the deletions of the recombinant adenoviral vector, toobtain recombinant adenovirus particles, infecting a culture of a secondtype of complementing cells with said recombinant adenovirus particles,wherein said second type of complementing cells express protein fromsaid first region of the adenovirus genome, but not protein from saidsecond region of the adenovirus genome, to produce the protein ofinterest; and harvesting the protein of interest.
 17. A method forproducing a protein of interest, the method comprising: infecting aculture of complementing cells with a recombinant adenoviral particlethat has a genome comprising nucleic acid encoding the protein ofinterest under control of a promoter to produce the protein of interest,wherein said genome has deletions in a first region and in a secondregion, and each of the first and second regions is required foradenoviral genome replication and/or adenovirus particle formation, andwherein said complementing cells express protein from said first regionof the genome of the adenoviral particle, but not protein from saidsecond region of the genome of adenoviral particle, and harvesting theprotein of interest.
 18. The method according to claim 17, wherein saidfirst region is E1.
 19. A method for producing a protein of interest,the method comprising: infecting a culture of complementing cells with arecombinant adenoviral particle that has a genome comprising nucleicacid encoding the protein of interest under control of a promoter toproduce the protein of interest, wherein said genome has deletions in afirst region and in a second region, and each of the first and secondregions is required for adenoviral genome replication and/or adenovirusparticle formation, and wherein said complementing cells express proteinfrom said first region of the genome of the adenoviral particle, but notprotein from said second region of the genome of adenoviral particle,and harvesting the protein of interest, wherein said first region is Eland wherein said second region is E2A.
 20. The method according to claim17, wherein said complementing cells Comprise PER.C6 cells as depositedat the ECACC under number 96022940.